Scaffolds for neural tissue and uses thereof

ABSTRACT

The present invention provides tissue scaffolds, methods of generating such scaffolds, and methods of use of such scaffolds to generate aligned and functional neural tissues for use in methods including regenerative medicine, wound repair and transplantation.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority under 35 U.S.C.365(c) and is also a Continuation-in-Part under 35 U.S.C. 120 of U.S.patent application Ser. No. 14/875,168, filed on Oct. 5, 2015, whichclaims priority under 35 U.S.C. 119(e) of U.S. Provisional ApplicationNo. 62/060,421, filed on Oct. 6, 2014. The former application is also aContinuation-in-Part under 35 U.S.C. 120 of U.S. patent application Ser.No. 14/363,331, filed on Jun. 6, 2014, which is the U.S. National Phaseof International Application No. PCT/US2012/068187, filed on Dec. 6,2012, which claims the benefit of priority of U.S. ProvisionalApplication No. 61/567,744, filed on Dec. 7, 2011. The disclosures ofall of the above applications are incorporated herein by reference intheir entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with U.S. government support under grant numbersCHE-0612572, CHE-0924104 and DMR-0819860 awarded by the National ScienceFoundation and grant numbers CA044627, CA160611 and GM059383 awarded bythe National Institutes of Health. The U.S. government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Tissue formation, wound repair, and many disease processes depend onexpression and cell-mediated assembly of the appropriate extracellularmatrix (ECM) proteins. In particular, oriented ECM fibers are essentialfor normal tissue development and homeostasis. The organization of theECM can, however, go awry in many diseases and at sites of injuryproducing the unaligned collagen fibers that form in scar tissue.

A goal of regenerative medicine is to promote formation of new tissuethat closely resembles the normal tissue in organization and function.Controlling cell growth in a spatially defined way enables regenerationof damaged or diseased tissues having the proper alignment ofconstituent cells and/or alignment of molecular complexes that the cellsproduce. In particular, cells direct the arrangement of ECM fibrils tocorrespond to their actin filaments by using cell surface receptors thatare indirectly connected to the actin cytoskeleton. Therefore, a majorchallenge in regenerative medicine is to promote cells to assemble ECMfibrils, such as collagen, into particular orientations or alignments ona scaffold device in order to generate tissues with the requiredfunctional properties.

Many methods for controlling cell growth rely on physical patterningprocedures that are not compatible with tissue scaffold deviceutilization. For example, grooved patterns that physically restraincells may affect cell functions, and stamping of biologics is size andthickness limited.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofmethods for generating cell-adhesive chemical (i.e., non-biologic)patterns in nano- and micro-scale dimensions using a technique forsurface-modifying solids or polymers to generate devices that haveutility as bio-scaffold materials and other devices including electrodesand sensors.

The devices of the present invention can be used in, for example,regenerative medicine, wound repair, and transplant biology, as well asin screening assays to determine the effects of a test compound onliving tissue by examining the effect of the test compound on variousbiological responses, such as for example, cell viability, cell growth,migration, differentiation and maintenance of cell phenotype.

Accordingly, in one aspect, the present invention provides patternedscaffolds for tissue. In one embodiment, the tissue scaffold comprises abase layer comprising a pattern of stripes, and an oxide layercomprising the pattern of stripes. In one embodiment the base layer andthe oxide layer comprise a pattern of oxide layer stripes on the baselayer.

In another embodiment, the present invention provides a tissue scaffoldcomprising a base layer comprising a pattern of stripes; an oxide layercomprising the pattern of stripes; and an extracellular matrix componentaligned in parallel on the stripes.

In another embodiment, the tissue scaffold comprises a base layercomprising a pattern of stripes, an oxide layer comprising the patternof stripes, and a non-biologic cell adhesive layer disposed on the oxidelayer.

In another embodiment, the present invention provides a tissue scaffoldcomprising a base layer comprising a pattern of stripes; an oxide layercomprising the pattern of stripes; a non-biologic cell adhesive layerdisposed on the oxide layer; and an extracellular matrix componentaligned in parallel with the stripes.

In another embodiment, the present invention provides an artificialtissue comprising living cells attached to a tissue scaffold comprisinga base layer comprising a pattern of stripes, and an oxide layercomprising the pattern of stripes.

In another embodiment, the present invention provides an artificialtissue comprising living cells attached to a tissue scaffold comprisinga base layer comprising a pattern of stripes; an oxide layer comprisingthe pattern of stripes; and a non-biologic cell adhesive layer disposedon the oxide layer.

In another embodiment, the present invention provides an artificialtissue comprising living cells attached to a tissue scaffold comprisinga base layer comprising a pattern of stripes; an oxide layer comprisingthe pattern of stripes; and an extracellular matrix component aligned inparallel with the stripes.

In another embodiment, the present invention provides an artificialtissue comprising living cells attached to a tissue scaffold comprisinga base layer comprising a pattern of stripes; an oxide layer comprisingthe pattern of stripes; a non-biologic cell adhesive layer disposed onthe oxide layer; and an extracellular matrix component aligned inparallel with the stripes.

In another embodiment, the present invention provides a method formaking a tissue scaffold comprising: generating a pattern of stripes ona base layer by photolithography to form a substrate having a patternedbase layer; and depositing an oxide layer onto the patterned base layerto form a patterned oxide layer. In another embodiment, the method formaking a tissue scaffold further comprises contacting the patternedoxide layer with a non-biologic cell adhesive compound to generate apatterned cell adhesive layer.

In another embodiment, the present invention provides a method formaking a tissue scaffold comprising: generating a pattern of stripes ona base layer by photolithography to form a substrate having a patternedbase layer; depositing an oxide layer onto the patterned base layer toform a substrate having a patterned oxide layer; contact the substratehaving the patterned oxide layer with cells and culturing underconditions suitable for the production of extracellular matrixcomponents; and removing the cells from the substrate to provide atissue scaffold comprising an extracellular matrix component aligned inparallel with the stripes.

In another embodiment, the present invention provides a method formaking a tissue scaffold comprising: generating a pattern of stripes ona base layer by photolithography to form a substrate having a patternedbase layer; depositing an oxide layer onto the patterned base layer toform a substrate having a patterned oxide layer; contacting thepatterned oxide layer with a non-biologic cell adhesive compound togenerate a substrate having a patterned cell adhesive layer; contactingthe substrate having the patterned cell adhesive layer with cells andculturing under conditions suitable for the production of extracellularmatrix components; and removing the cells from the substrate to providea tissue scaffold comprising an extracellular matrix component alignedin parallel with the stripes.

In another embodiment, the present invention provides a method formaking an artificial tissue comprising living cells attached to a tissuescaffold comprising contacting a tissue scaffold of the presentinvention with cells and culturing under conditions suitable for cellgrowth and/or differentiation.

In other embodiments, the present invention provides medical devicescomprising the artificial tissues or tissue scaffolds of the invention.

In other embodiments, the present invention provides methods of tissuerepair and regeneration comprising implanting the artificial tissues ofthe present invention in a subject in need of such tissue repair orregeneration.

In another embodiment, the present invention provides methods foridentifying a compound that modulates a tissue function. The methodsinclude providing a patterned scaffold for tissue as described herein,contacting the tissue with a test compound, and measuring the effect ofthe test compound on a tissue function in the presence and absence ofthe test compound, wherein a modulation of the tissue function in thepresence of the test compound as compared to the tissue function in theabsence of the test compound indicates that the test compound modulatesa tissue function, thereby identifying a compound that modulates atissue function.

In another embodiment, the present invention provides methods foridentifying a compound useful for treating or preventing a tissuedisease. The methods include providing a patterned scaffold for tissueas described herein, contacting the tissue with a test compound, andmeasuring the effect of the test compound on a tissue function in thepresence and absence of the test compound, wherein a modulation of thetissue function in the presence of the test compound as compared to thetissue function in the absence of the test compound indicates that thetest compound modulates a tissue function, thereby identifying acompound useful for treating or preventing a tissue disease.

In another embodiment, scaffolds for nerve tissue regeneration andmethods of use thereof are provided that overcome the limitations ofprevious systems and methods for controlling cell alignment on polymersubstrates by using cell-assembled, aligned ECM patterned polymerscaffolding that controls neurite outgrowth as opposed to theunpatterned scaffolding systems of previous inventions. The presence ofaligned ECM patterns in the scaffolding systems of the present inventionhelps promote and coordinate neurite growth in the directions of thealigned ECM patterns. In specific embodiments of the tissue scaffold thecells are neural cells. The neural cells can be selected from the groupconsisting of neural stem cells, oligopotent stem cells, differentiatedneurons, differentiated glial cells and neural crest cells. In someembodiments the tissue scaffold containing neural cells is co-culturedwith glial support cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are schematics of the preparation of ananoscale-patterned surface using a self-assembled monolayer ofphosphonate (SAMP). 1A. (A) Spin-cast with hexamethyldisilazane (HMDS);(B) spin-cast AZ-5412E photoresist; (C) expose to UV through aphotomask; (D) develop in AZ-312 MIF; (E) vapor deposition of zirconiumtetra-t-butoxide (1), then heat to form adhesion layer; (F) assembly ofthe SAMP. 1B. The photolithographically patterned surface (E) is treatedwith vapor of 1 and then heated to give 2; reaction with phosphonic acid3 gives SAMP/ZrO2/surface 4.

FIGS. 2A, 2B and 2C depict box plots for cell aspect ratio definedaccording to (A) and measured for (B) and angle of orientation (C).Plots represent the distribution of all measurements (n=100) for cellaspect ratio and angle of orientation. The boxes span 25-75 percentilesfor the distributions; the whiskers span the 5-95 percentiles. Thesquare is at the average value, and the horizontal line is at themedian. The asterisk (*) denotes no statistical difference compared tothe control.

FIGS. 3A, 3B, 3C and 3D show NIH 3T3 cells spread on SAMP/ZrO2/SiO2/Siafter 24 hr: (A) 20×10, (B) 30×30, (C) 100×40, (D) control. Scalebars=100 Actin filaments and nuclei are shown. Insets show conformity ofcell attachment with the SAMP/ZrO₂ patterns (the lighter stripes).

FIG. 4 illustrates intensities of the radial sums as a function ofangle, which represents cell conformity with the SAMP/ZrO2/SiO2/Sipattern. These plots correspond to the images in FIGS. 3A-D. Full-widthat half-maximum value (FWHM) for 20×10, 32°; 30×30, 29°; 100×40, 49°;FWHM for the control is not applicable. The peaks narrow as thedimensions decrease, indicating greater conformity of the cell to thepattern.

FIGS. 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H and 5I show 3T3 fibroblasts on30×30 (top), 10×10 (middle) SAMP/ZrO2/SiO2/Si patterned surfaces andunpatterned control surfaces (bottom) after 24 hours, three days, andeight days. Images A, D and G after 24 hr; B, E, and H after 3 days; C,F, and I after eight days. As shown, 3T3 cells grow to confluence butremain in line with the chemical pattern. Images show actin stain; scalebar=100 μm; patterned directions in A-F are not identical with regard tothe page.

FIGS. 6A, 6B, 6C, 6D, 6E and 6F show bone marrow derived hMSCs on 30×30SAMP/ZrO2/SiO2/Si patterned surfaces (A-C) and control, unpatternedsurfaces (D-F). Images A and D after 24 hr, B and E after 3 days, and Cand F after 8 days. As shown, hMSCs align with the chemical pattern andremain in alignment as they grow to confluence over 8 days; nodirectionality was observed for the unpatterned, control surfaces. Scalebars=100 μm; patterned directions in A-C are not identical with regardto the page.

FIGS. 7A, 7B, 7C, 7D, 7E, 7F, 7G, 7H and 7I show SEM images(Magnification=200×) of 30×30 SAMP/ZrO2 on Nylon (A), polyethyleneterephthalate (PET; D), polyetheretherketone (PEEK; G). As shown, thepattern is uniform across the polymer surface, with well-defined edges.3T3 Fibroblasts on patterned Nylon (B), PET (E), and PEEK (H) after 24hr, and Nylon (C), PET (F), and PEEK (I) after 3 days. Cell images showactin stain, scale bar=100 μm; patterned directions in A-I are notidentical with regard to the page.

FIG. 8 shows phase contrast images of NIH3T3 cells growing on patternedand unpatterned PET.

FIG. 9 shows fibronectin alignment on PET surfaces and results of fastFourier transformation (FFT) analysis of the images of the cellsconfirming fibronectin alignment on the patterned PET surfaces.

FIG. 10 shows the results of fast Fourier transformation analysis offibronectin alignment on patterns of different dimensions.

FIG. 11 shows collagen alignment on PET surfaces and results of fastFourier transformation analysis of the images of the cells confirmingcollagen alignment on the patterned PET surfaces.

FIG. 12 shows extracellular matrix on patterned PET surfaces before andafter decellularization, fibronectin (FN) alignment and collagen type I(Col-I) alignment after decellularization, and a scanning electronmicrograph (SEM) of the entire matrix. The bar graph and graph show thatangular alignment of collagen on the pattern is good, and that there isessentially no directionality of collagen on the unpatterned surface.

FIG. 13 shows neurites from plated PC12 cells on decellularized matrixon unpatterned (top) and patterned (bottom) surfaces. As shown, neuritesaligned in the direction of collagen fibril orientation on patternedsurfaces, and there was no alignment on the unpatterned control.

FIG. 14 shows fibroblast alignment after one day on nylon, PET and PEEKsurfaces treated with ZrO2 and ZrO2/SAMP.

FIG. 15 shows fibroblast alignment after three days on nylon, PET andPEEK surfaces treated with ZrO2 and ZrO2/SAMP.

FIG. 16 represents the process by which the micropatterned polymerscaffolding base is generated, utilizing basic photolithographictechniques.

FIGS. 17A, 17B and 17C represent fibroblast alignment on themicropatterned polymer scaffolding base. NIH3T3 cells cultured onunpatterned (a) or a 10×10 μm striped pattern (b, c) were visualized byphase contrast microscopy after 4 hours (a, b) and 10 days (c). Scalebars=100 μm. Double arrow indicates the orientation of the ZrO2/SAMPpattern (in b) and the ends of several pattern stripes are indicated byasterisks (*, in b).

FIGS. 18A, 18B, 18C, 18D, 18E and 18F represent oriented assembly of ECMcomprised of fibronectin and collagen fibrils. NIH 3T3 fibroblasts weregrown to confluence on unpatterned (a) or 10×10 μm patterned (b, e)ZrO2/SAMP-modified PET substrates. The matrix was detected on day 10 byimmunostaining with anti-fibronectin antiserum (a, b) or anti-type Icollagen antibodies (e). Scale bars=100 μm. Double arrows indicatepattern orientation. Images were processed by two-dimensional FFT, andthe radial summation of pixel distributions around the origin in the FFToutputs was plotted between 0° and 180° (c, f). The FWHM of each pixelintensity curve in (c) was calculated and is plotted in (d). Col-I: typeI collagen; FN: fibronectin. **p<0.0005.

FIGS. 19A, 19B, 19C, 19D, 19E and 19F represent the characterization ofdecellularized ECM. After decellularization matrices were stained withanti-fibronectin (a) or anti-collagen type I antibodies (b) and the FFTpixel intensity distribution plots for fibronectin (FN) and type Icollagen (Col-I) in this matrix are shown in (c). (d, e) Images of typeI collagen fibrils in decellularized matrix on unpatterned or 10×10 μmZrO2/SAMP patterned PET were used for FFT analysis (d) and the FWHM ofthese curves were determined (e). (f) SEM image of decellularizedmatrix. Scale bars=100 μm (a, b) and 2 μm (f). *p<0.05.

FIGS. 20A, 20B, 20C, 20D, 20E and 20F represent directional neuriteoutgrowth on decellularized ECM. PC12 cells were induced to extendneurites with NGF for 72 hr on unpatterned (a, c) or 10×10 μm patterned(b, d) decellularized matrix PET substrates. Cells were visualized bystaining the actin cytoskeleton with rhodamine-phalloidin (a-d; red inc, d). Matrix fibrils were immunostained with anticollagen type Iantibodies (c, d; green). Double arrow indicates pattern orientation.Scale bars=100 μm. (e, f) Angles of neurites were measured relative toan arbitrary orientation for unpatterned (e) or to the pattern stripes(f) and binned in ranges of 20° centered on 90° which is parallel to thepattern. Each histogram of the angular distributions of neurites wasfitted to a Gaussian to calculate the FWHM values.

FIG. 21 represents directional radial glial cell alignment and growth.GFP-tagged radial glial cells were cultured for 2 and 4 days on ECM andwas then plotted as a histogram to observe angular alignment with theECM.

FIG. 22 represents how PC12 cells and glial cells align with ECM PC12cells were differentiated with neural growth factor (NGF) and culturedfor 3 days on decellularized ECM and stained for F-actin to observeneurite alignment. Radial glial cells were initially cultured for 2 dayson ECM and PC12 cells were then seeded onto substrate and co-culturedfor 3 days. FFTs were used to determine alignment of cells with FWHMhighlighted for cells on unpatterned and patterned ECM.

FIGS. 23A and 23B demonstrate neurite outgrowth and directionality usingsympathetic cervical ganglia. Substrates included a glass coverslipcoated with a 10 μg/ml solution of laminin (Laminin), decellularizedfibroblast ECM on an unpatterned glass coverslip (Unpatterned ECM), anddecellularized aligned ECM on a 20 μm×20 μm patterned PET surface(Patterned ECM). A single ganglion with extended neurites is shown ineach image (FIGS. 23A and 23B). The substrate is indicated above theimage. Scale bar=500 μm. In FIG. 23A neurite outgrowth was fitted withan ellipse (lines on images) to determine if neurite extension deviatedfrom circular. The aspect ratios (long axis divided by the short axis)displayed in the graph were calculated and averaged for a minimum of 3samples per condition. The asterisk (*) indicates that the aspect ratioon patterned ECM is significantly different from the other substrates.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofmethods for generating cell-adhesive chemical (i.e., non-biologic)patterns in nano- and micro-scale dimensions using a technique forsurface-modifying solids or polymers to prepare devices that haveutility as bio-scaffold materials, electrodes, sensors or other devices.

The devices and methods of the present invention are simpler thanpreviously described methods and devices in that it is not necessary toprovide a biologic cell adhesion molecule, such as an RGD peptide, tomimic an in vivo environment to properly align and/or orient cells toform a functional tissue in vitro with appropriate cellular organizationand biological activities. In other words, it is not necessary tofunctionalize the chemical layer to elicit a biological response using amaterial such as a saccharide, an oligosaccharide, a polysaccharide, anucleic acid, a protein and/or a peptide. The lack of biologiccell-adhesive materials prevents encumbrance of cell receptors that maybe involved in cell spreading and extracellular interactions affectingcellular organization and tissue alignment. Further, the patterns do notaffect the physical properties of the substrate. The devices and methodsof making the devices of the present invention are also simpler thanpreviously described methods and devices in that they can be performedin about one hour and the base layer used to generate the scaffolds doesnot need to comprise a reactive side-chain-containing species.

In the devices and methods of the present invention, the patterns aremore cell-adhesive than the substrates on which the patterns aredeveloped. Cells attach to the patterns but are not constrained into thepatterns by physical means. As the cells proliferate, they align intounmodified areas of the patterns to form confluent monolayers of cellswhile maintaining pattern alignment across the substrate surface.

Accordingly, described herein are methods and devices which may be usedin a broad range of applications, for example, in regenerative medicine,wound repair, transplant biology, drug delivery, testing the effect ofsubstances upon cells, tissue formation, cell actuation, anddevelopmental biology.

Furthermore, the methods described herein are amenable to a wide varietyof hard or soft surfaces, including soft polymeric surfaces.

I. Devices of the Invention and Methods of Production of the Same

Accordingly, in one aspect, the present invention provides patternedscaffolds for tissue. In one embodiment, the present invention providesa tissue scaffold comprising a base layer comprising a pattern ofstripes, and an oxide layer comprising the pattern of stripes. Inanother embodiment, the tissue scaffold comprises a base layercomprising a pattern of stripes, an oxide layer comprising the patternof stripes, and a non-biologic cell adhesive layer disposed on the oxidelayer.

A base layer for use in the present invention may be a solid, rigid, orhard polymeric surface, a semi-rigid polymeric surface, a soft polymericsurface, a hard non-polymeric surface, a semi-rigid non-polymericsurface, or a soft non-polymeric surface. In, one embodiment, a baselayer is biologically inert. In one embodiment, a base layer comprisestwo or more surfaces. For example, a base layer comprising a softpolymeric or non-polymeric surface may be placed temporarily on a solidor semi-rigid polymeric or non-polymeric surface.

In some embodiments, a base layer for use in the compositions andmethods of the invention may have a Young's modulus of about 0.001-0.1,0.005-0.2, 0.005-0.5, 0.05-1.0, 0.075-1.0, 0.1-2.0, 1.0-2.0, 1.5-5.0,2.0-5.0, 3.0-7.0, 3.0-10, 5.0-15, 5.0-20, 10-20, 15-30, 20-30, 25-50,30-50, 50-75, 50-100, 75-125, 100-150, 125-150, 150-200, 175-200,200-250, or about 250-300 gigapascals (GPa). Ranges and valuesintermediate to the above recited ranges and values are alsocontemplated to be part of the invention. For example, a Young's modulusof about 6.5-9.8, or 5.2-7.8 GPa is intended to be encompassed by thepresent invention.

In other embodiments, a base layer for use in the compositions andmethods of the invention may have a Young's modulus of about 0.001-0.1,0.005-0.2, 0.005-0.5, 0.05-1.0, 0.075-1.0, 0.1-2.0, 1.0-2.0, 1.5-5.0,2.0-5.0, 3.0-7.0, 3.0-10, 5.0-15, 5.0-20, 10-20, 15-30, 20-30, 25-50,30-50, 50-75, 50-100, 75-125, 100-150, 125-150, 150-200, 175-200,200-250, or about 250-300 kilopascals (kPa). Ranges and valuesintermediate to the above recited ranges and values are alsocontemplated to be part of the invention. For example, a Young's modulusof about 6.5-9.8, or 5.2-7.8 kPa is intended to be encompassed by thepresent invention. For nerve regeneration, a lower Young's modulus maybe used.

In one embodiment, the base layer is selected from the group consistingof a hard polymeric surface, a semi-rigid polymeric surface, and a softpolymeric surface, a hard non-polymeric surface, a semi-rigidnon-polymeric surface, and a soft non-polymeric surface. In oneembodiment, the base layer comprises a polyamide, a polyamide hydrogel,a polyurethane, a polyurea, a polyester, a polyester hydrogel, apolyketone, a polyimide, a polysulfide, a polysulfoxide, a polysulfone,a polythiophene, a polypyridine, a polypyrrole, polyethers, silicone(polysiloxane), polysaccharides, fluoropolymers, epoxies, aramides,amides, imides, polypeptides, polyethylene, polystyrene, polypropylene,glass reinforced epoxies, liquid crystal polymers, thermoplastics,bismaleimide-triazine (BT) resins, benzocyclobutene ABFGx13, lowcoefficient of thermal expansion (CTE) films of glass and epoxies,polyvinyls, polyacrylics, polyacrylates, polycarbonates,polytetrafluoroethylene (PTFE), polyethylene terephthalate (PET),quartz, silicon (e.g., silicon wafers), glass, ceramic, metals and metalalloys including titanium, titanium alloys, tantalum, zirconium,stainless steel and cobalt-chromium alloys, metal oxides, poly(vinylpyrrolidone), poly(-hydroxyethyl methacrylate), poly(N-vinylpyrrolidone), poly(methyl methacrylate), poly(vinyl alcohol),poly(acrylic acid), polyacrylamide, polyacrylamide hydrogels,polyrethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylicacid), polylactides (PLA), polyglycolides (PGA),poly(lactide-co-glycolides) (PLGA), polylactones, polylactone hydrogels,polyanhydrides, polyphosphazenes, polygermanes, polyorthoesters,polyolefins, poly-carbonates, biopolymers, such as a silk, collagen,copolymers and derivatives thereof, and composites including thesepolymers. In one preferred embodiment, the base layer is silicon,polyetheretherketone, nylon, including nylon 6,6 or PET. In anotherpreferred embodiment, the base layer is silicone or polyacrylamide. Inother preferred embodiments the base layer is hydrophilicpolyacrylamide, hydrophilic polyester, polyacrylamide hydrogel,polyester hydrogel or polylactone hydrogel. In another preferredembodiment, the base layer is silk or collagen. In another preferredembodiment, the base layer is titanium or stainless steel.

In one embodiment, the base layer is a support structure.

In one embodiment, the support structure is selected from the groupconsisting of a Petri dish, a cover-slip, a glass slide, a multi-wellplate, a microfluidic chamber, an implant, and a medical device.

Medical devices for use as base layers in the present invention include,for example, diagnostic implant devices, biosensors, stimulators, neuralstimulators, neural activity recorders, diabetic implants such asglucose monitoring devices, external fixation devices, external fixationimplants, orthopedic trauma implants, implants for use in joint andspinal disorders/reconstruction such as plates, screws, rods, plugs,cages, scaffolds, artificial joints (e.g., hand, wrist, elbow, shoulder,spine, hip, knee, ankle), wires and the like, oncology related bone andsoft tissue replacement devices, dental and oral/maxillo-facial devices,cardiovascular implants such as stents, catheters, valves, rings,implantable defibrillators, and the like, contact lenses, ocularimplants, keratoprostheses, dermatologic implants, cosmetic implants,implantable medication delivery pumps; general surgery devices andimplants such as but not limited to drainage catheters, shunts, tapes,meshes, ropes, cables, wires, sutures, skin staples, burn sheets, andvascular patches; and temporary or non-permanent implants.

The base layer comprises a pattern of stripes having at least twoparallel stripes wherein adjacent stripes are separated by a space. Inone embodiment, the width and spacing of the stripes is about 0.1 μm toabout 1000 μm.

The width and spacing of the stripes may be varied over the range fromabout 0.1 μm to about 1000 μm, from about 1 μm to about 500 μm, fromabout 1 μm to 250 μm, from about 1 μm to 160 μm, from about 1 μm to 100μm, from about 1 μm to 90 μm, from about 1 μm to 80 μm, from about 1 μmto 70 μm, from about 1 μm to 60 μm, from about 1 μm to 50 μm, from about1 μm to 40 μm, from about 1 μm to 30 μm, from about 1 μm to 20 μm, fromabout 1 μm to 10 μm about 2 μm to 100 μm, from about 2 μm to 90 μm, fromabout 2 μm to 80 μm, from about 2 μm to 70 μm, from about 2 μm to 60 μm,from about 2 μm to 50 μm, from about 2 μm to 40 μm, from about 2 μm to30 μm, from about 2 μm to 20 μm, from about 2 μm to 10 μm, from about 1μm to 100 μm, from about 5 μm to about 160 μm, from about 5 μm to about100 μm, from about 5 μm to about 90 μm, from about 5 μm to about 80 μm,from about 5 μm to about 70 μm, from about 5 μm to about 60 μm, fromabout 5 μm to about 50 μm, from about 5 μm to about 40 μm, from about 5μm to about 30 μm, from about 5 μm to about 20 μm, and from about 5 μmto about 10 μm. Ranges and values intermediate to the above recitedranges and values are also contemplated to be part of the invention. Forexample, a width and spacing of about 4-28, or 7-19 μm are intended tobe encompassed by the present invention.

The width and spacing of the stripes can be equivalent or different. Forexample, both the width and spacing can be about 0.1, about 0.2, about0.25, about 0.5, about 0.75, about 1, about 2, about 3, about 4, about5, about 6, about 7, about 8, about 9, about 10, about 11, about 12,about 13, about 14, about 5, about 16, about 17, about 18, about 19, orabout 20 μm. In other embodiments, the width can be about 0.1, about0.2, about 0.25, about 0.5, about 0.75, about 1, about 2, about 3, about4, about 5, about 6, about 7, about 8, about 9, about 10 μm, about 11,about 12, about 13, about 14, about 15, about 16, about 17, about 18,about 19, about 20, about 21, about 22, about 23, about 24, about 25,about 26, about 27, about 28, about 29, about 30, about 31, about 32,about 33, about 34, about 35, about 36, about 37, about 38, about 39, orabout 40 μm, and the spacing can be about 1, about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10, about 11, about12, about 13, about 14, about 15, about 16, about 17, about 18, about19, about 20, about 21, about 22, about 23, about 24, about 25, about26, about 27, about 28, about 29, about 30, about 31, about 32, about33, about 34, about 35, about 36, about 37, about 38, about 39, or about40 μm. Values intermediate to the above recited values are alsocontemplated to be part of the invention. For example, a width andspacing of about 5.6 or 23.1 μm are intended to be encompassed by thepresent invention.

In one preferred embodiment, the stripes are about 5 μm to 100 μm wideand spaced about 5 μm to 100 μm apart. In another preferred embodiment,the stripes are about 5 μm wide and spaced about 5 μm apart. In anotherpreferred embodiment, the stripes are about 10 μm wide and spaced about10 μm apart. In another preferred embodiment, the stripes are about 20μm wide and spaced about 20 μm apart. In yet another preferredembodiment, the stripes are about 30 μm wide and spaced about 30 μmapart. In yet another preferred embodiment, the stripes are about 20 μmwide and spaced about 10 μm apart.

An oxide for use in the oxide layer of the scaffold on the presentinvention includes any compound(s) that when contacted with thepatterned base layer forms a continuous layer on the patterned baselayer. In one embodiment, the oxide is a metal oxide, e.g., formed froman alkoxide precursor. In one embodiment the alkoxide is of a transitionmetal. Periodic Table Group 3-6 and 13-14 metals are transition metalssuitable for use in the present invention. Such metals include Zr, Al,Ti, Hf, Ta, Nb, V and Sn. Depending upon the position of the transitionmetal on the Periodic Table, a transition metal alkoxide will have fromthree to six alkoxide groups or a mixture of oxo and alkoxide groups. Inone embodiment, an alkoxide group has from 2 to 4 carbon atoms, andincludes, for example, ethoxide, propoxide, iso-propoxide, butoxide,iso-butoxide, tert-butoxide and fluoronated alkoxide. In one embodiment,a metal alkoxide for use in the present invention is zirconiumtetra(tert-butoxide). In another embodiment, a metal alkoxide for use inthe present invention is tantalum pentaethoxide. Suitable oxides for usein the present invention also include those described in U.S. PatentPublication No. 2009/0104474 and PCT Publication No. WO 2009/052352, theentire contents of each of which are incorporated herein by reference.In one preferred embodiment, the oxide layer comprises a metal oxide. Inanother preferred embodiment, the metal oxide is formed from theprecursor zirconium tetra(tert-butoxide).

For embodiments in which a non-biologic cell adhesive layer is disposedon the oxide layer, the cell adhesive layer comprises an adhesivechemical compound. A cell adhesive chemical compound is any organiccompound that is sufficiently reactive to react with the oxide layer,e.g., sufficiently reactive with a metal oxide or alkoxide, such as, forexample, an organic compound comprising a phosphonic, carboxylic,sulfonic, phosphinic, phosphoric, sulfinic, or hydroxamic acid group. Inone preferred embodiment, the cell adhesive layer comprises aphosphonate.

In certain embodiments, the scaffolds further comprise living cells. Ithas been discovered in accordance with the present invention that cellscan adhere to the patterned oxide layers in the absence of a celladhesive layer disposed on the oxide layer.

Accordingly, in one embodiment, the present invention provides anartificial tissue comprising living cells attached to a tissue scaffoldcomprising a base layer comprising a pattern of stripes, and an oxidelayer comprising the pattern of stripes. In another embodiment, thepresent invention provides an artificial tissue comprising living cellsattached to a tissue scaffold comprising: a base layer comprising apattern of stripes; an oxide layer comprising the pattern of stripes;and a non-biologic cell adhesive layer disposed on the oxide layer. Thetype of cells is not limited, and includes, for example, fibroblasts,endothelial cells, keratinocytes, osteoblasts, chondroblasts andchondrocytes, hepatocytes, macrophages, cardiac muscle cells, smoothmuscle cells, skeletal muscle cells, tendon cells, ligament cells,neural cells, epithelial cells, and stem cells. Stem cells includeembryonic stem cells, adult stem cells, and induced pluripotent stemcells. In one preferred embodiment, the cells are mesenchymal stemcells. In another preferred embodiment, the cells are human cells.

In another embodiment, the present invention provides a tissue scaffoldcomprising: a base layer comprising a pattern of stripes; an oxide layercomprising the pattern of stripes; and an extracellular matrix componentaligned in parallel with the stripes. In another embodiment, the presentinvention provides a tissue scaffold comprising: a base layer comprisinga pattern of stripes; an oxide layer comprising the pattern of stripes;a non-biologic cell adhesive layer disposed on the oxide layer; and anextracellular matrix component aligned in parallel with the stripes.Extracellular matrix components are known in the art and include forexample, fibronectin and collagens.

The scaffolds comprising an extracellular matrix component may furthercomprise living cells attached to the matrix component. Accordingly, inanother embodiment, the present invention provides an artificial tissuecomprising living cells attached to a tissue scaffold comprising: a baselayer comprising a pattern of stripes; an oxide layer comprising thepattern of stripes; and an extracellular matrix component aligned inparallel with the stripes. In another embodiment, the present inventionprovides an artificial tissue comprising living cells attached to atissue scaffold comprising: a base layer comprising a pattern ofstripes; an oxide layer comprising the pattern of stripes; anon-biologic cell adhesive layer disposed on the oxide layer; and anextracellular matrix component aligned in parallel with the stripes. Thetype of cells is not limited, and includes, for example, fibroblasts,endothelial cells, keratinocytes, osteoblasts, chondroblasts andchondrocytes, hepatocytes, macrophages, cardiac muscle cells, smoothmuscle cells, skeletal muscle cells, tendon cells, ligament cells,neural cells, epithelial cells, and stem cells. Stem cells includeembryonic stem cells, adult stem cells, and induced pluripotent stemcells. In one preferred embodiment, the cells are mesenchymal stemcells. In another preferred embodiment, the cells are human cells. Thecells need not be the same cells used to produce the extracellularmatrix component on the substrate. The cells may be obtained from asubject to be treated with the artificial tissue generated on thesubstrate.

In another embodiment, the present invention provides a method formaking a tissue scaffold comprising: generating a pattern of stripes ona base layer by photolithography to form a patterned base layer; anddepositing an oxide layer onto the patterned base layer to form apatterned oxide layer. In another embodiment, the method for making atissue scaffold further comprises contacting the patterned oxide layerwith a non-biologic cell adhesive compound to generate a patterned celladhesive layer.

Methods of photolithography known in the art may be used to form thepatterned base layer. For example, the method may comprise depositing aphotoresist onto the base layer, thereby generating a photoresist layer,placing a mask on top of the photoresist layer and exposing thephotoresist layer to ultraviolet radiation, thereby generating apatterned base layer.

As used herein, the term “depositing” refers to a process of placing orapplying an item or substance onto another item or substance (which maybe identical to, similar to, or dissimilar to the first item orsubstance). Depositing may include, but is not limited to, methods ofusing spraying, dip casting, spin coating, evaporative methods, sputtermethods, immersion methods, extractive deposition methods, or othermethods to associate the items or substances. The term depositingincludes applying the item or substance to substantially the entiresurface as well as applying the item or substance to a portion of thesurface.

In one embodiment, spin coating is used to apply a photoresist layeronto the base layer. Spin coating is a process wherein the base layeris, for example, mounted to a chuck under vacuum and is rotated to spinthe base layer about its axis of symmetry and a liquid or semi-liquidsubstance, e.g., a photoresist, is dripped onto the base layer, with thecentrifugal force generated by the spin causing the liquid orsemi-liquid substance to spread substantially evenly across the surfaceof the base layer. Variations of this process, for example coating andthen spinning, or spinning and then dripping, may also be used.

“Photoresist” is any substance that is sensitive to ultravioletradiation, e.g., wave-lengths of light in the ultraviolet or shorterspectrum (<400 nm). A photoresist may be positive or negative. Suitablephotoresists include, without limitation, AZ-5412E, AZ-701, AZ-1505,AZ-1518, and AZ-4330 (all are available from AZ Electronic Materials).In one embodiment the photoresist is AZ-5412E.

A base layer comprising a photoresist may be patterned by providing amask comprising the desired shape and/or pattern, i.e., a stripedpattern. The mask may be a solid mask such as a photolithographic mask.The mask is provided and placed on top of the photoresist layer.Subsequently, a portion of the photoresist layer (i.e., the portion ofthe photoresist not covered by the mask) is exposed to ultravioletradiation.

The mask placed on top of the photoresist layer is typically fabricatedby standard photolithographic procedure, e.g., by means of electron beamlithography. Other methods for creating such masks include focusedenergy for ablation (micromachining) including lasers, electron beamsand focused ion beams. Similarly, chemical etchants may be used to erodematerials through the photoresist when using an alternative maskmaterial. Examples of chemical etchants include hydrofluoric acid andhydrochloric acid. Photo-lithographic masks are also commerciallyavailable.

Any suitable material, e.g., a material that has a flat surface, e.g., ametal (gold, silver, platinum, tantalum, or aluminum), a ceramic(alumina, titanium oxide, silica, or silicon nitride), may be used formaking the mask.

In some embodiments, a combination of positive and negative photoresistscan be used. For example, a positive photoresist is deposited on a baselayer in a particular pattern and subsequently a negative photoresist ina complementary pattern is applied. This results in a patterned tissuescaffold that comprises a pattern that comprises regions that are morecell adhesive next to regions that are less cell-adhesive.

Once the photoresist layer is exposed to ultraviolet radiation and apatterned base layer is formed, the mask is removed and an oxide isdeposited to the patterned base layer to form a patterned oxide layer.The oxide binds directly onto the base layer and does not depend on theintroduction of reactive side chain-containing species into thepolymeric base layer.

One embodiment of the invention encompasses a method for making a tissuescaffold comprising the steps of generating a pattern on a base layer ofprotective photoresist compound stripes and unprotected base layerstripes using photolithography, to form a patterned base layer; anddepositing an oxide layer onto the unprotected stripes to form apatterned oxide layer.

In one embodiment, a thin oxide layer, e.g., a metal oxide, is depositedonto the patterned base layer as a continuous layer. As used herein, theterm “continuous layer” is a layer that is formed by a matrix ofindividual molecules that are chemically bonded and linked to eachother, as opposed to individual molecules covering the surface. In thepresent case, in one embodiment, oxide molecules, e.g., metal oxidemolecules, are bonded together on at least a portion of the patternedbase layer to form a continuous layer. In another embodiment, a thinoxide layer, e.g., a metal oxide, is deposited onto the patterned baselayer as a non-continuous layer, i.e., a pattern of individual moleculescovering the surface.

An oxide for use in the oxide layer of the scaffold on the presentinvention includes any compound(s) that when contacted with thepatterned base layer forms a continuous layer on the patterned baselayer. In one embodiment, the oxide is a metal oxide, e.g., formed froman alkoxide precursor. In one embodiment the alkoxide is of a transitionmetal. Periodic Table Group 3-6 and 13-14 metals are transition metalssuitable for use in the present invention. Such metals include Zr, Al,Ti, Hf, Ta, Nb, V and Sn. Depending upon the position of the transitionmetal on the Periodic Table, a transition metal alkoxide will have fromthree to six alkoxide groups or a mixture of oxo and alkoxide groups. Inone embodiment, an alkoxide group has from 2 to 4 carbon atoms, andincludes, for example, ethoxide, propoxide, iso-propoxide, butoxide,iso-butoxide, tert-butoxide and fluoronated alkoxide. In one embodiment,a metal alkoxide for use in the present invention is zirconiumtetra(tert-butoxide). In another embodiment, a metal alkoxide for use inthe present invention is tantalum pentaethoxide. Suitable oxides for usein the present invention also include those described in U.S. PatentPublication No. 2009/0104474 and WO 09/052352, the entire contents ofeach of which are incorporated herein by reference. In one preferredembodiment, the oxide layer comprises a metal oxide. In anotherpreferred embodiment, the metal oxide is formed from the precursorzirconium tetra(tert-butoxide).

The oxide is deposited onto the patterned base layer under conditionssuitable to form an oxide layer on the patterned base layer. This may beachieved using any suitable technique known to one of ordinary skill inthe art and includes, for example, vapor or immersion deposition andsol-gel processes. The step of forming a patterned oxide layer mayinclude subjecting the oxide to pyrolysis, microwaving, completehydrolysis or partial hydrolysis. In one embodiment, when heatingconditions are employed, the oxide is heated to between about 50° C. andabout the melting point of the polymer, e.g., not at or above themelting point of the polymer. In another embodiment, when heatingconditions are employed, the oxide is heated to between about 50° C. andabout the glass transition temperature of the polymer, e.g., not at orabove the glass transition temperature of the polymer.

In a preferred embodiment, the oxide is deposited by vapor phasedeposition of a metal alkoxide. In another preferred embodiment, themetal alkoxide is zirconium tetra(tert-butoxide).

The thickness of the patterned chemical layer is controlled by thedeposition and heating times. Shorter exposure times for deposition(about 5 minutes) and heat (about 10 minutes) generally produce about a1 nm layer (about 2 monolayers). The thickness of the layer can bedetermined by, for example, quartz crystal microgravimetry (QCM).

In an embodiment, the patterned chemical layer is about 0.1 to about 100nm, 0.1 to about 70 nm, about 0.1 to about 50 nm, about 0.1 to about 30nm, 0.1 to about 20 nm, about 0.1 to about 10 nm, is about 0.1 to about10 nm, 0.1 to about 7 nm, about 0.1 to about 5 nm, about 0.1 to about 3nm, 0.1 to about 2 nm, about 0.1 to about 1 nm, about 0.5 to about 2 nm,about 1 to about 2 nm, about 1 to about 1.5 nm, about 1.5 to about 2 nm,or about 0.1 nm, 0.5 nm, 1 nm, 1.1 nm, 1.2 nm, 1.3 nm, 1.4 nm, 1.5 nm,1.6 nm, 1.7 nm, 1.8 nm, 1.9 nm, 2 nm, 2.1 nm, 2.2 nm, 2.3 nm, 2.4 nm,2.5 nm, 2.6 nm, 2.7 nm, 2.8 nm, 2.9 nm, 3.0 nm, 3.1 nm, 3.2 nm, 3.3 nm,3.4 nm, 3.5 nm, 3.6 nm, 3.7 nm, 3.8 nm, 3.9 nm, 4.0 nm, 4.1 nm, 4.2 nm,4.3 nm, 4.4 nm, 4.5 nm, 4.6 nm, 4.7 nm, 4.8 nm, 4.9 nm, 5.0 nm, 5.1 nm,5.2 nm, 5.3 nm, 5.4 nm, 5.5 nm, 5.6 nm, 5.7 nm, 5.8 nm, 5.9 nm, 6.0 nm,6.1 nm, 6.2 nm, 6.3 nm, 6.4 nm, 6.5 nm, 6.6 nm, 6.7 nm, 6.8 nm, 6.9 nm,7.0 nm, 7.1 nm, 7.2 nm, 7.3 nm, 7.4 nm, 7.5 nm, 7.6 nm, 7.7 nm, 7.8 nm,7.9 nm, 8.0 nm, 8.1 nm, 8.2 nm, 8.3 nm, 8.4 nm, 8.5 nm, 8.6 nm, 8.7 nm,8.8 nm, 8.9 nm, 9.0 nm, 9.1 nm, 9.2 nm, 9.3 nm, 9.4 nm, 9.5 nm, 3.6 nm,9.7 nm, 9.8 nm, 9.9 nm, or about 10.0 nm in thickness. In anotherembodiment, the patterned chemical layer is 2 nm or less in thickness.In an embodiment, the patterned chemical layer is about 1 to about 1.5nm in thickness. In an embodiment, the patterned chemical layer is about10 to about 70 nm in thickness. In another embodiment, multiple layersof a semi-rigid or soft polymer are coated on the base layer so long asthe polymer can still flex. It should be understood that a range betweenany two figures listed above is specifically contemplated to beencompassed within the metes and bounds of the present invention.

The patterned oxide layer can be about 1 to about 50, about 1 to about45, about 1 to about 40, about 1 to about 35, about 1 to about 30, about1 to about 25, about 1 to about 20, about 1 to about 15, about 1 toabout 10, about 1 to about 5, about 2 to about 50, about 2 to about 45,about 2 to about 40, about 2 to about 35, about 2 to about 30, about 2to about 25, about 2 to about 20, about 2 to about 15, about 2 to about10, about 2 to about 5, about 5 to about 50, about 5 to about 45, about5 to about 40, about 5 to about 35, about 5 to about 30, about 5 toabout 25, about 5 to about 20, about 5 to about 15, about 5 to about 10,about 10 to about 50, about 10 to about 45, about 10 to about 40, about10 to about 35, about 10 to about 30, about 10 to about 25, about 10 toabout 20, about 10 to about 15, about 20 to about 50, about 25 to about50, about 30 to about 50, about 35 to about 50, about 40 to about 50, orabout 45 to about 50 monolayers thick. Ranges and values intermediate tothe above recited ranges and values are also contemplated to be part ofthe invention. For example, 1-3 and 7-11 monolayer thicknesses areintended to be encompassed by the present invention.

In one embodiment, the patterned oxide layer is about 1 to about 10,about 1 to about 9, about 1 to about 8, about 1 to about 7, about 1 toabout 6, about 1 to about 5, about 1 to about 4, about 1 to about 3,about 1 to about 2 monolayers thick. In one embodiment, the patternedoxide layer is more than about 2 monolayers thick. In anotherembodiment, the patterned oxide layer less than about 2 monolayersthick. In another embodiment, the patterned oxide layer about 1monolayer thick.

For making a scaffold that further comprises a cell adhesive layer, acell adhesive chemical compound is deposited onto the patterned oxidelayer, to form a patterned cell adhesive layer. The cell adhesivechemical compound is any organic compound that is sufficiently reactivewith the oxide layer, e.g., sufficiently reactive with a metal oxide oralkoxide, such as, for example, an organic compound comprising aphosphonic, carboxylic, sulfonic, phosphinic, phosphoric, sulfinic, orhydroxamic group. Any suitable method may be used to deposit the organiclayer onto the patterned oxide layer. In one embodiment, the celladhesive chemical compound is deposited onto the patterned oxide layerby a dipping method.

In one embodiment, the patterned oxide layer is contacted with aphosphonic acid to generate a patterned cell adhesive layer comprising aphosphonate. As used herein, the term “phosphonic acid” refers to agroup, or compound that includes a group of formula —P(═O)(OH)2 attachedto a carbon atom, As used herein, the term “phosphonate,” refers to agroup, or compound that includes a group of formula —P(═O)(OX)2 attacheddirectly to a carbon atom where X is the metal from the patterned oxidelayer.

In one embodiment, the acid used to treat the patterned oxide layercomprises any aliphatic or aromatic moiety and contains two acidicgroups. In one embodiment the acidic groups are phosphonic acid groups(—P(═O)(OH)2). In another embodiment the acidic groups are carboxylicacid groups (—CO2H). In yet another embodiment the acidic groups areboth phosphonic and carboxylic acid groups. In one embodiment the acidused to treat the patterned oxide layer comprises an alkyl chain. In oneembodiment the alkyl chain is 4-18 carbons in length. In anotherembodiment the alkyl chain is optionally substituted with any aromaticor aliphatic moiety, e.g. an alkyl, aryl, alkenyl or alkynyl group.Non-limiting examples of phosphonic acids include α,ω-bisphosphonicacids. Non-limiting examples of carboxylic acids includeα,ω-biscarboxylic acids. Nonlimiting examples of mixedphosphonic/carboxylic acids include α-carboxylic-ω-phosphonic acids.Non-limiting examples of mixed phosphonic/carboxylic acids includeα-phosphonic-ω-carboxylic acids. In one embodiment, the bisphosphonicacid is an octadecylphosphonic acid (ODPA) derivative. In oneembodiment, the phosponic acid is 1,4-butane-diphosphonic acid.

In some embodiments, a cell-avoidance layer is deposited on all or aportion of the patterned oxide layer. In one embodiment, acell-avoidance layer may be deposited in a pattern that is complementaryto a cell adhesive layer. A cell-avoidance layer is a layer thatinhibits the adhesion of cells.

Exemplary compounds suitable for use as a cell-avoidance layer include,for example, compounds with terminal pegylated groups and thosecomprising an alkyl terminal group.

In certain embodiments, the scaffolds of the present invention furthercomprise living cells. Accordingly, in another embodiment, the presentinvention provides a method for making an artificial tissue comprisingliving cells attached to a tissue scaffold comprising contacting atissue scaffold of the present invention with cells and culturing underconditions suitable for cell growth and/or differentiation. The type ofcells is not limited, and includes, for example, fibro-blasts,endothelial cells, keratinocytes, osteoblasts, chondroblasts andchondrocytes, hepatocytes, macrophages, cardiac muscle cells, smoothmuscle cells, skeletal muscle cells, tendon cells, ligament cells,neural cells, epithelial cells, and stem cells. Stem cells includeembryonic stem cells, adult stem cells, and induced pluripotent stemcells. In one preferred embodiment, the cells are mesenchymal stemcells. In another preferred embodiment, the cells are human cells.Culture conditions for cell growth and/or differentiation are known tothose of skill in the art.

As noted above, one embodiment of the invention encompasses a method formaking a tissue scaffold comprising the steps of (1) generating apattern on a base layer of protective photoresist compound stripes andunprotected base layer stripes using photolithography, to form apatterned base layer; and (2) depositing an oxide layer onto theunprotected stripes to form a patterned oxide layer. One embodimentfurther comprises contacting the patterned oxide layer of the substratewith cells; culturing the cells under conditions suitable for theproduction of extracellular matrix components; and removing the cellsfrom the substrate to provide a tissue scaffold comprising anextracellular matrix component aligned on the stripes.

In certain embodiments, the scaffolds of the present invention furthercomprise an extracellular matrix component aligned in parallel with thepattern of stripes. Accordingly, in another embodiment, the presentinvention provides a method for making a tissue scaffold comprisinggenerating a pattern of stripes on a base layer by photolithography toform a substrate having patterned base layer; depositing an oxide layeronto the patterned base layer to form a substrate having a patternedoxide layer; contacting the substrate having the patterned oxide layerwith cells and culturing under conditions suitable for the production ofextracellular matrix components; and removing the cells from thesubstrate to provide a tissue scaffold comprising an extracellularmatrix component aligned in parallel with the stripes.

In yet another embodiment, the present invention provides a method formaking a tissue scaffold comprising generating a pattern of stripes on abase layer by photolithography to form a substrate having patterned baselayer; depositing an oxide layer onto the patterned base layer to form asubstrate having a patterned oxide layer; contacting the patterned oxidelayer with a non-biologic cell adhesive compound to generate a substratehaving a patterned cell adhesive layer; contacting the substrate havingthe patterned cell adhesive layer with cells and culturing underconditions suitable for the production of extracellular matrixcomponents; and removing the cells from the substrate to provide atissue scaffold comprising an extracellular matrix component aligned inparallel with the stripes. Thus, one embodiment of the method furthercomprises contacting the patterned cell adhesive layer of the substratewith cells; culturing the cells under conditions suitable for theproduction of extracellular matrix components; and removing the cellsfrom the substrate to provide a tissue scaffold comprising anextracellular matrix component aligned on the stripes.

In the two foregoing embodiments, the patterned substrates comprisingcells may be made by the methods described hereinabove, and thencultured under conditions suitable for the production of extracellularmatrix components. Extracellular matrix components include, for example,fibronectin and collagens. Such conditions are known to those of skillin the art. These conditions include culturing cells at sufficientdensity in the presence of growth factors or serum. The skilled artisancan optimize conditions for different cell types. Additives may be usedto stimulate the production of certain proteins. For example, type Icollagen production may be stimulated by adding ascorbic acid to theculture medium.

After production of extracellular matrix components, the cells may beremoved from the substrate, i.e., the substrate is decellularized.Methods for decellularizing are known in the art and include, forexample, methods to loosen cell attachments from the extracellularmatrix followed by lysis of cell membranes and solubilization ofintracellular components under conditions that maintain the integrityand activity of the matrix. For example, decellularization may beaccomplished by treatment to remove calcium by chelation to loosen cellattachments, followed by incubation with non-ionic detergent in ahypotonic buffer at alkaline pH to lyse cell membranes and solubilizeintracellular components.

The scaffolds comprising an extracellular matrix component may furthercomprise living cells attached to the matrix component. Accordingly, inanother embodiment, the present invention provides a method for makingan artificial tissue comprising living cells attached to a tissuescaffold comprising the step of contacting a tissue scaffold of thepresent invention with cells and culturing under conditions suitable forcell growth and/or differentiation. The type of cells is not limited,and includes, for example, fibroblasts, endothelial cells,keratinocytes, osteoblasts, chondroblasts and chondrocytes, hepatocytes,macrophages, cardiac muscle cells, smooth muscle cells, skeletal musclecells, tendon cells, ligament cells, neural cells, epithelial cells, andstem cells. Stem cells include embryonic stem cells, adult stem cells,and induced pluripotent stem cells. In one preferred embodiment, thecells are mesenchymal stem cells. In another preferred embodiment, thecells are human cells. The cells need not be the same cells used toproduce the extracellular matrix component on the substrate. The cellsmay be obtained from a subject to be treated with the artificial tissuegenerated on the substrate. Culture conditions for cell growth and/ordifferentiation are known to those of skill in the art.

For example, cells may be attached by placing the scaffold in culturewith a cell suspension and allowing the cells to settle and adhere tothe surface. Cells respond to the patterning in terms of adherence andin terms of assembling ECM proteins in the pattern on the scaffold.Cells also respond to the patterning in terms of maturation, growth andfunction. The cells on the scaffold may be cultured in an incubatorunder physiologic conditions (e.g., at 37° C.) until the cells form atwo-dimensional (2D) tissue, the orientation of which is determined bythe pattern provided on the tissue scaffold.

Any appropriate cell culture method may be used to establish the tissueon the tissue scaffold. The seeding density of the cells will varydepending on the cell size and cell type but can easily be determined bymethods known in the art. In one embodiment, cells are seeded at adensity of between about 1×10³ to about 6×10⁵ cells/cm², or at a densityof about 1×10³, about 2×10³, about 3×10³, about 4×10³, about 5×10³,about 6×10³, about 7×10³, about 8×10³, about 9×10³, about 1×10⁴, about2×10⁴, about 3×10⁴, about 4×10⁴, about 5×10⁴, about 6×10⁴, about 7×10⁴,about 8×10⁴, about 9×10⁴, about 1×10⁵, about 1.5×10⁵, about 2×10⁵, about2.5×10⁵, about 3×10⁵, about 3.5×10⁵, about 4×10⁵, about 4.5×10⁵, about5×10⁵, about 5.5×10⁵, about 6×10⁵, about 6.5×10⁵, about 7×10⁵, about7.5×10⁵, about 8×10⁵, about 8.5×10⁵, about 9×10⁵, about 9.5×10⁵, about1×10⁶, about 1.5×10⁶, about 2×10⁶, about 2.5×10⁶, about 3×10⁶, about3.5×10⁶, about 4×10⁶, about 4.5×10⁶, about 5×10⁶, about 5.5×10⁶, about6×10⁶, about 6.5×10⁶, about 7×10⁶, about 7.5×10⁶, about 8×10⁶, about8.5×10⁶, about 9×10⁶, or about 9.5×10⁶ cells/cm². Values and rangesintermediate to the above-recited values and ranges are alsocontemplated by the present invention.

In one embodiment of the invention, the patterned tissue scaffold iscontacted with a plurality of cells and cultured such that a livingtissue, e.g., a tissue having at least in part, in vivo biologicalactivity, is produced. In one embodiment, a living tissue is removedfrom the tissue scaffold. In one embodiment the patterned tissuescaffold comprises a plurality of cells on the stripes, and can beorganized into a tissue structure. The cells can comprise human cells.Further the cells can consist essentially of a member selected from thegroup consisting of fibroblasts, endothelial cells, keratinocytes,osteoblasts, chondroblasts, chondrocytes, hepatocytes, macrophages,cardiac muscle cells, smooth muscle cells, skeletal muscle cells, tendoncells, ligament cells, neural cells, epithelial cells, and stem cells.

II. Exemplary Uses of the Patterned Tissue Scaffolds of the Invention

The patterned tissue scaffolds of the invention (and/or a living tissueprepared on a tissue scaffold and removed from the scaffold) may be usedin a broad range of applications, including, but not limited to, devicesfor use in tissue repair and support such as sutures, surgical andorthopedic screws, and surgical and orthopedic plates, natural coatingsor components for synthetic implants, cosmetic implants and supports,repair or structural support for organs or tissues, substance delivery,bioengineering platforms, platforms for testing the effect of substancesupon cells, cell culture, wound healing, neural regeneration andnumerous other uses. In one embodiment, the living tissue is removedfrom the scaffold prior to use. In another embodiment, the living tissueis not removed from the scaffold prior to use.

The base layer of the patterned tissue scaffolds of the presentinvention may be tissue prepared on the tissue scaffolds of theinvention or a medical device, such as an orthopedic screw or plate thatcomprises cells of the same tissue in which the devices will be used.Non-limiting examples of medical devices suitable for use in the presentinvention include, diagnostic implant devices, biosensors, stimulators,diabetic implants such as glucose monitoring devices, external fixationdevices, external fixation implants, orthopedic trauma implants,implants for use in joint and spinal disorders/reconstruction such asplates, screws, rods, plugs, cages, scaffolds, artificial joints (e.g.,hand, wrist, elbow, shoulder, spine, hip, knee, ankle), wires and thelike, oncology related bone and soft tissue replacement devices, dentaland oral/maxillofacial devices, cardiovascular implants such as stents,catheters, valves, rings, implantable defibrillators, and the like,contact lenses, ocular implants, keratoprostheses, dermatologicimplants, cosmetic implants, implantable medication delivery pumps;general surgery devices and implants such as but not limited to drainagecatheters, shunts, tapes, meshes, ropes, cables, wires, sutures, skinstaples, burn sheets, and vascular patches; and temporary/non-permanentimplants.

In addition, because the methods described herein are applicable to softpolymeric base layers, flexible membranes comprising a patterned tissuescaffold may be used to support or connect tissue or structures thathave experienced injury, surgery, or deterioration. For example, apatterned tissue scaffold comprising a soft polymer may be used as agraft to connect and/or bind tissue and provide a platform for tissueregeneration, internally or externally. In such instances, the softpolymer may be biodegradable or non-biodegradable.

Another exemplary use of the patterned soft tissue scaffolds of theinvention is as a barrier for the prevention of post-operative inducedadhesion(s). For example, because adhesions are the result ofdisorganized ECM, the patterned tissue scaffolds of the invention may beused to organize ECM deposition and prevent the formation of adhesions.

Yet another exemplary use of the patterned tissue scaffolds of theinvention is as templates for nerve growth. For example, the patternedtissue scaffolds may be used to culture neural cells in a patternmimicking the in vivo environment such that suitable neural connectionsform rather than the unorganized array of neural cells that are producedwithout use of a patterned scaffold. The patterned scaffolds areprepared according to FIG. 16.

The components of the nervous system are categorized as belonging toeither the peripheral nervous system (PNS) or the central nervous system(CNS). The basic anatomy of the nervous system at a cellular level isdefined by the presence of neurons, specialized cells which areelectrically excitable and serve to transmit information throughelectrical and chemical signaling. Signaling between neurons occurs viasynapses, which are membrane-to-membrane junctions between neuron cells,or between neurons and other cell tissues, for example between efferentnerve fibers and muscle fibers at the neuromuscular junction.

Aside from neurons, the other primary cellular component of the nervoussystem is glial cells. Glial cells are non-neuronal cells which providesupport for neurons in a variety of manners, including maintaininghomeostasis, providing nutrients and oxygen, providing structuralintegrity to the neural network, providing and maintaining a myelinsheath, and to combat pathogens and remove dead neurons. It is alsopossible that glial cells may assist neurons to form synapticconnections with other neurons.

The CNS is the part of the nervous system that is comprised of the brainand the spinal cord. The PNS is the part of the nervous system that iscomprised of nerves that are outside the CNS, in that the PNS excludesthe brain and the spinal cord. The PNS is primarily comprised ofganglia, which are clusters of neurons found in the PNS. Ganglia in thePNS are comprised mainly of somata and dendritic structures which areinterconnected to other ganglia to form a system known as a plexus.Ganglia primarily function to provide intermediate connections betweendifferent neurological structures in the body, including differentstructures in the PNS, and between the PNS and the CNS. There are threemajor groups of ganglia found among vertebrate animals, which includethe dorsal root ganglia, the cranial nerve ganglia, and the autonomicganglia.

Neurons are comprised of a number of different structure features. At abasic level, neurons are comprised of dendrites, a soma (cell body)containing the nucleus and other essential cellular machinery, an axon,a long thin nerve fiber that conducts electrical impulses away from thedendrites to the axon terminal, which is oriented to another neuron'sdendrites or to a target cellular tissue. A major characteristic ofneurons is the presence or absence of a myelin sheath along the axon ofthe neuron. Neurons may be either myelinated or unmyelinated, dependingon the location of the neuron in the nervous system. Myelin is anelectrically insulating, i.e. dielectric material primarily comprised oflipids, proteins, cholesterol and water, the precise compositions ofeach varying by its location in the nervous system. The myelin sheathserves to increase the speed at which electrical impulses are conductedalong the axon. In the PNS, Schwann cells (glial cells) provide themyelin sheath, whereas in the CNS, oligodendrocytes provide the sheath.

A defining characteristic of neurons is that once neural progenitorcells have fully differentiated into neurons, the neurons do not undergoany further cellular division. Instead, new neurons arise via neuralstem cell differentiation.

Accordingly, in one embodiment, this invention relates to scaffolds fornerve tissue regeneration. In another embodiment, this invention relatesto methods for regenerating nerve tissue. In general, the presentinvention provides methods of tissue repair and regeneration comprisingimplanting the artificial tissues of the present invention in a subjectin need of such tissue repair or regeneration. Another embodiment isdirected to a method of tissue repair or regeneration comprisingimplanting a tissue scaffold of invention into tissue in need of repairor regeneration in a subject in need thereof.

In one embodiment, neural cells seeded onto the patterned polymerscaffold will grow in alignment with the pattern on the polymer.

In another embodiment, once the decellularized, cell-assembled, alignedECM patterned polymer scaffolding has been created according to themethods disclosed herein, neural cells may be seeded onto thescaffolding. The neural cells will grow in alignment with the fibrillarmatrix. Quantification of neurite expansion has shown enhanced alignmentof neural cells when cultured on patterned/aligned ECM compared to anunpatterned matrix. By controlling the initial alignment of the cellsthat synthesize the ECM, and in effect the ECM alignment, thescaffolding can be rendered bioactive to more precisely control andregulate nerve tissue growth, differentiation, and regeneration.

The neural cells which are seeded on the scaffolding can be induced toconnect or reconnect with other neural cells, or potentially othercellular targets, for example, but not limited to, muscle tissue at theNMJ. This has wide potential therapeutic application as a treatment fora number of neurodegenerative diseases as well as aging.

There are a large number of cell types that are of potential use asneural cells. Some non-limiting examples include stem cells, includingbut not limited to, omnipotent, pluripotent, and induced pluripotentstem cells, for example, embryonic stem cells and adult stem cellsincluding, but not limited to, neural stem cells (NSCs), as well asoligopotent stem cells including, but not limited to, neural progenitorcells and neuroblasts. Co-culture with glial support cells is anotherpossibility, including, but not limited to CNS macroglial cells, forexample, astrocytes and astrocyte precursor cells, oligodendrocytes andoligodendrocyte precursor cells, and PNS macroglial cells, for exampleSchwann cells and Schwann precursor cells. Further possible cell typesinclude already differentiated neurons and glial cells, neural crestcells, such as those that give rise to the PNS, including, but notlimited to, the dorsal root ganglia, and neural tube cells, such asthose that give rise to the CNS, including, but not limited to, thebasal ganglia.

Furthermore, there are a number of commercially available suitable cellculture lines for use with the present invention. These include, but arenot limited to, the PC12/PC12 Adh cell line, derived frompheochromocytoma of the rate adrenal medulla. Other appropriate celllines include, but are not limited to, those derived from neuroblasts,including, but not limited to, Neuro-2a, N1E-115, NB41A3, B104-1-1,which are cell lines derived from mice neuroblasts, and SK-N-AS, IMR-32,SK-N-DZ, SK-N-FI, BE(2)-M17, BE(2)-C, which are cell lines derived fromhuman neuroblasts. Further appropriate cell lines include, but are notlimited to, those derived from Schwann cell lines, includingimmortalized Schwann cell lines, for example, but not limited to, SW10(mouse), R3 and RSC96 (rat) neuronal Schwann cells. One having ordinaryskill in the art will appreciate that these cell lines listed are merelyexemplary and many numerous other potential cell lines are appropriatefor use with the current invention.

In some embodiments, patterned tissue scaffolds contacted or seeded withliving cells are combined with a drug such that the function of animplant or graft will improve. For example, antibiotics,anti-inflammatories, local anesthetics or combinations thereof, can beadded to the cell-treated a patterned tissue scaffold to speed thehealing process. Other compounds, molecules, elements, or ions such asgrowth factors, trophic factors, cytokines, steroids, metabolites, andother signaling molecules and hormones such as those involved in growthand differentiation, for example, but not limited to, signalingmolecules involved in neural development and differentiation, such asthose signaling molecules involved in the Notch signaling pathway, maybe added to the scaffolding in order to promote, direct, alter, orrepress axon alignment, outgrowth, and neural regeneration and cellulargrowth.

In one embodiment, the tissues of the present invention can be used tostudy functional differentiation of stem cells (e.g., pluripotent stemcells, multipotent stem cells, induced pluripotent stem cells, andprogenitor cells of embryonic, fetal, neonatal, juvenile and adultorigin). For example, the patterned tissue scaffolds of the inventionare contacted with undifferentiated cells, e.g., stem cells, anddifferentiation is observed.

Patterned tissue scaffolds containing cells are also useful formeasuring tissue activities or functions, investigating tissuedevelopmental biology and disease pathology, as well as in drugdiscovery.

Accordingly, the present invention also provides methods for identifyinga compound that modulates a tissue function. The methods includeproviding a tissue scaffold comprising a tissue produced according tothe methods of the invention, contacting the tissue with a testcompound; and measuring the effect of the test compound on a tissuefunction in the presence and absence of the test compound, wherein amodulation of the tissue function in the presence of the test compoundas compared to the tissue function in the absence of the test compoundindicates that the test compound modulates a tissue function, therebyidentifying a compound that modulates a tissue function.

In another aspect, the present invention also provides methods foridentifying a compound useful for treating or preventing a disease. Themethods include providing a tissue scaffold comprising a tissue producedaccording to the methods of the invention, contacting a tissue with atest compound; and measuring the effect of the test compound on a tissuefunction in the presence and absence of the test compound, wherein amodulation of the tissue function in the presence of the test compoundas compared to the tissue function in the absence of the test compoundindicates that the test compound modulates a tissue function, therebyidentifying a compound useful for treating or preventing a disease.

PRESENT EMBODIMENTS

One embodiment of the invention is directed to a tissue scaffoldcomprising a base layer and an oxide layer, where the base layer and theoxide layer comprise a pattern of oxide layer stripes on the base layer,and the tissue scaffold further comprises a plurality of neural cells onthe stripes, where the neural cells are selected from the groupconsisting of neural stem cells, oligopotent stem cells, differentiatedneurons, differentiated glial cells and neural crest cells. Oneembodiment of the tissue scaffold further comprises a non-biologic celladhesive layer disposed on the oxide layer. Another embodiment furthercomprises an extracellular matrix component aligned on the stripes. Inone embodiment of the tissue scaffold the base layer is selected fromthe group consisting of a semi-rigid polymeric surface and a softpolymeric surface. In another embodiment the base layer is selected fromthe group consisting of polyesters, polyamides, polylactones,polyacrylamides and polysiloxanes. In yet another embodiment the baselayer is selected from the group consisting of silk and collagen. Instill another embodiment the base layer is selected from the groupconsisting of nylon, polyethylene terephthalate, polycaprolactonefumarate, polyethylene glycol fumarate, and methylene bis acrylamide.

In one embodiment of the tissue scaffold the stripes are about 5 μm to100 μm wide and spaced about 5 μm to 100 μm apart. In some embodimentsthe stripes are about 5 μm wide and spaced about 5 μm apart, or about 10μm wide and spaced about 10 μm apart, or about 20 μm wide and spacedabout 20 μm apart, or about 30 μm wide and spaced about 30 μm apart, orabout 20 μm wide and spaced about 10 μm apart.

In one embodiment of the tissue scaffold the oxide is a metal oxide. Inone embodiment the metal oxide is zirconium oxide.

In some embodiments of the tissue scaffold the cell adhesive layercomprises a phosphonate.

In some embodiments of the tissue scaffold the oligopotent stem cellsare selected from the group consisting of neural progenitor cells andneuroblasts.

In some embodiments of the tissue scaffold the neural crest cells areselected from the group consisting of dorsal root ganglia cells, neuraltube cells, and basal ganglia cells.

In some embodiments the tissue scaffold is co-cultured with glialsupport cells. In some embodiments the glial support cells are selectedfrom the group consisting of CNS macroglial cells and PNS macroglialcells. In some embodiments the CNS macroglial cells are selected fromthe group consisting of astrocytes, astrocyte precursor cells,oligodendrocytes and oligodendrocyte precursor cells. In someembodiments the PNS macroglial cells are selected from the groupconsisting of Schwann cells and Schwann precursor cells.

As used herein, the various forms of the term “modulate” are intended toinclude stimulation (e.g., increasing or upregulating a particularresponse or activity) and inhibition (e.g., decreasing or downregulatinga particular response or activity).

As used herein, the term “contacting” (e.g., contacting a tissue with atest compound) is intended to include any form of interaction (e.g.,direct or indirect interaction) of a test compound and a tissue. Theterm “contacting” includes incubating a compound and a tissue (e.g.,adding the test compound to a tissue).

Test compounds, may be any agents including chemical agents (such astoxins), small molecules, pharmaceuticals, peptides, proteins (such asantibodies, cytokines, enzymes, and the like), and nucleic acids,including gene medicines and introduced genes, which may encodetherapeutic agents, such as proteins, antisense agents (i.e., nucleicacids comprising a sequence complementary to a target RNA expressed in atarget cell type, such as RNAi or siRNA), ribozymes, and the like.

The test compound may be added to a tissue by any suitable means. Forexample, the test compound may be added drop-wise onto the surface of atissue of the invention and allowed to diffuse into or otherwise enterthe tissue, or it can be added to the nutrient medium and allowed todiffuse through the medium.

All references cited herein are incorporated herein in their entireties.

The following examples serve to further illustrate the presentinvention.

Example 1 Materials and Methods

Materials: p-Type, heavily boron-doped silicon terminated with a 1000 Åthermally grown oxide layer (Silicon Quest, Inc.), polyetheretherketone,nylon 6,6, and polyethylene terephthalate films of 0.05 mm thickness(Goodfellow, Corp.), glass cover slips (12 mm, No. 2; VWR) were obtainedfrom commercial sources. The dimensions of the photolithographicpatterns used were (width of stripe X space between stripes; all nominaldimensions are in μm): 10×10, 20×20, 10×20, 20×10, 20×30, 30×10, 30×30,40×30, 50×30, 60×30, and 100×40. Some minor shadowing was observed withstripes≤20 μm. Hexanes, toluene, methanol, 2-propanol,hexamethyldisilazane (HMDS), formaldehyde, 4′,6-diamidino-2-phenylindole(DAPI), and anti-vinculin antibodies (Sigma-Aldrich); zirconiumtetra(tert-butoxide) (Strem Chemicals, Inc.); fluorescein-taggedsecondary antibody, Dulbecco's modified eagle medium (DMEM)(Invitrogen); bone marrow derived human mesenchymal stem cells (hMSCs,PT-2501), serum containing “bullet” medium (PT-3001), trypsin/EDTA(CC-3232) (Lonza); nonyl phenoxypolyethoxylethanol (NP-40), sulfuric andhydrochloric acids (EMD Chemicals); 30% hydrogen peroxide (J. T. Baker);rhodamine phalloidin (Molecular Probes); 1,4-butanediphosphonic acid(Acros Organics); AZ-5214E photoresist and AZ-312 MIF developer (CapitolScientific, Inc.); absolute ethyl alcohol (Pharmco-Aaper) were used asreceived. Photomasks were fabricated using a Heidelburg DWL 66 laserwriter equipped with a 20 mm focal length writehead. NIH 3T3 mousefibroblasts and bone marrow derived hMSCs were passaged bi-weekly andwere stored at 37° C. until use.

Cleaning of Substrates: Silicon wafers were cut into 1 cm×1 cm coupons;coupons and glass cover slips were cleaned by sequential sonication inhexanes, toluene, and methanol for 15 min. The samples were nextimmersed in “piranha” solution (H₂SO₄: 30% H₂O₂, 3:1) for 15 min at 85°C. followed by sequential rinsing in deionized water and 2-propanol, andthen dried under a stream of nitrogen. A second acid cleaning was donein “buzzard” solution (HCl: 30% H₂O₂, 1:1) at 85° C. for 15 min; thesamples were rinsed sequentially with deionized water and 2-propanol,and then dried under a stream of nitrogen. The cleaned surfaces werestored in a desiccator until use. Polymer films were cut into 1 cm×1 cmcoupons and were cleaned by sonication in ethanol for 15 min. Thesubstrates were rinsed with 2-propanol, dried under a stream ofnitrogen, and stored in a desiccator until use.

Photolithography: Neither specialized equipment nor a clean room arerequired. Cleaned polymer and silicon surfaces were rinsed sequentiallywith ethanol and 2-propanol, then dried under nitrogen and finallyheated (95° C.) for 10 min, with the exception of nylon and PET, whichwere not heated to avoid glass transition. HMDS was spin cast onto thesubstrate surfaces (4000 rpm, 40 sec) followed by AZ-5214E photoresist(4000 rpm, 40 sec). Substrates were baked for 45 sec (95° C.), exposedto UV (365 nm, 4 W) through a photomask for 30 sec, and then developedin AZ-312 MIF for 30-34 sec. The substrates were rinsed vigorously indeionized water and examined by optical microscopy. All patterns werefabricated and analyzed at minimum in duplicate.

Vapor Phase Deposition of Zirconium tetra(tert-butoxide) (1), andFormation of the self-assembled monolayer of phosphonate (SAMP):Substrates patterned with photoresist were placed inside a depositionchamber equipped with two valves; one was connected to vacuum and theother to a bulb containing zirconium tetra(tert-butoxide) (1). Thechamber was evacuated to 1×10⁻³ torr for 10 min. Samples were exposed tovapor of 1 for 3 min with the chamber opened to vacuum. The bulb andchamber were sealed, and the chamber was warmed to 50° C. for siliconand 75° C. for polymers, giving a cross-linked, zirconium oxide baselayer. The chamber was then cooled to room temperature. The chamber wasback-filled with zero-grade nitrogen, and valves were closed to isolatethe chamber prior to dismounting. The chamber was opened, and thesubstrates were soaked in an ethanol solution of 1,4-butanediphosphonicacid (0.25 mg/mL) for 24 hr. The preparation of the nanoscale-patternedsurface is depicted schematically in FIGS. 1A and 1B. The substrateswere then rinsed sequentially with ethanol and 2-propanol, dried undernitrogen, and then inspected by optical microscopy. This procedureremoves the HMDS, the photoresist, and any ZrO₂ on the photoresist toleave a negative pattern in which the SAMP/ZrO₂ is directly attached tothe substrate. The ability to make multiple, patterned substrates islimited only by the size of the photolithographic mask, the UV source,and the deposition chamber.

Surface Characterization: SAMP/ZrO₂ was analyzed by X-ray photoelectronspectroscopy (XPS), scanning electron microscopy (SEM), and energydispersion spectroscopy (EDS). A VG scientific ESCALab Mk II equippedwith a Mg Kα (1253.6 keV) anode source operating at 15 keV acceleratingvoltage and 20 mA and a VG scientific hemispherical sector analyzer(HAS) detector were used. A pass energy of 50 eV was used to collectsurvey (1000 to 0 eV) XPS data. Detailed XPS data were collected at apass energy of 20 eV with a dwell time of 500 μs and a step size of 0.05eV. Data analysis was carried out using CasaXPS software (Casa SoftwareLtd.). Spectra were calibrated against adventitious C 1s (at 284.5 eV).SEM analysis used a FEI Quanta 200 Environmental-SEM equipped with anOxford INCA Synergy 450 energy-dispersive X-ray microanalysis systemwith an X-Max 80 large area analytical silicon drift detector (SDD) atan acceleration voltage of 5 keV. Atomic force microscopy (AFM) was usedto determine the height on the patterns deposited on the substratecoupons (Digital Instruments Multimode AFM in tapping mode).

Pattern stability studies: Silicon and glass substrates werephotolithographically patterned with 30×30 stripes of ZrO₂ as describedabove. The substrates were immersed in serum-containing medium (DMEMwith 10% calf serum) for 18 days. Glass and Si substrates were removedon days 3, 6, 9, 12, and 18 and were rinsed gently with PBS. Opticalmicroscopy determined if the ZrO₂ stripes remained intact, XPS analysiswas used to identify elemental presence on the surface, and AFMinvestigated the surface morphology.

Cell alignment, toxicity, and long term studies: Cell adhesion studiesfor statistical analysis of cell aspect ratio and alignment wereconducted using NIH 3T3 fibroblasts. SAMP-Patterned and control(unpatterned SAMP) samples were placed in individual wells of 24-wellplates and were rinsed twice with phosphate buffered saline (PBS). NIH3T3 fibroblasts were plated at 30,000 cells per well on the substratesin serum-free DMEM and were allowed to attach at 37° C. for 3 hr. Themedium was changed to DMEM with 10% calf serum and the attached cellswere allowed to spread for an additional 21 hr (24 hr total). Cells werefixed using 3.7% formaldehyde in PBS for 15 min, permeabilized with 0.5%NP-40 in PBS for 15 min at room temperature, and stained withrhodamine-phalloidin and DAPI for cell shape and orientation studies.Images were captured using a Nikon TE2000U microscope.

Cell toxicity was studied using 3T3 cells plated on silicon surfacescompletely coated with a SAMP of 1,4-butanediphosphonic acid bonded ontoZrO₂ as described above. Cells were plated as described above induplicate, and were allowed to proliferate for 3 days; after 24 hrs and3 days they were stained and analyzed as above. Cells were counted andcell-spreading areas were measured using ImageJ, 10 image fields wereused for the measurements and field dimen-sions were 853 μm×683 μm. Celldoubling time was calculated assuming constant growth rate.

Long-term (8 day) cell studies were performed on silicon and polymerpatterned surfaces. 3T3 Cells were plated at 30,000 cells/well inserum-containing DMEM (10% calf serum). Bone marrow-derived MSCs weretrypsinized with trypsin/EDTA and plated at 30,000 cells/well in serumcontaining “bullet” medium. The cells were incubated at 37° C. as thecells grew to confluence. Time points on silicon were taken at 24 hr, 3days, and 8 days at which time the cells were stained for imaging asdescribed above. Polymer time points were 24 hr and 3 days. Cells wereplated on all surfaces in duplicate.

Data Analysis and Statistics: Cell conformity with SAMP/ZrO₂ patterns onSiO₂/Si surfaces was evaluated in fluorescent cell images by applyingthe Fourier transform using an ImageJ script (Oval Profile Plot scriptavailable at rsbweb.nih.gov/ij/index.html) (X. Fu, H. Wang, Tissue Eng.Pt. A 2012, 18, 631); images were rotated to center maximum intensitypeaks at 90°. Data were plotted using Origin 8.5 (OriginLab Ltd.) asintensity of radial sum versus angle. Peak fitting was used to obtainfull width at half maximum (FWHM) values for comparison. Data from 0° to180° are presented, three pictures were analyzed in each case, and theFWHM for each peak was averaged.

Cell aspect ratio was calculated using ImageJ and was defined as theratio of cell width to length; measurements were made after 24 hr onboth experimental and control surfaces. Long membrane projections thatwere ≤5.3 μm wide were not included in the measurement of the overalllength. The angle of cell orientation was also measured with respect tothat of the pattern for the experimental surfaces (see FIG. 2A). Anarbitrary horizontal axis was defined as the reference for the controlsurfaces; typically 5 fields were analyzed to measure 100 cells. TheShapiro-Wilk test was used to determine if data were normallydistributed (n=100, α<0.05). The Kruskal-Wallis one-way analysis ofvariance was used to test for significant difference between allexperimental groups and the control for both cell aspect ratio and angleof orientation (n=100, α<0.05). The Mann-Whitney U nonparametricstatistical hypothesis test was used to test for significance in apair-wise manner comparing the experimental groups, individually, to thecontrol; the Bonferoni correction was used to adjust the significancelevel (n=100, α<0.0045). Origin 8.5 software was used to generate boxplots.

Example 2 Preparation and Characterization of Surface-ModifiedSubstrates

Materials surfaces were chemically modified as shown in FIG. 1A.Spin-casting of HMDS was followed by spin-casting AZ-5412E photoresist;the photoresist-coated material was exposed to UV light through aphotomask of a negative of the desired striped pattern; the surface wasdeveloped in AZ-312 MIF to remove exposed photoresist; the substrate wasexposed to vapor of zirconium tetra(tert-butoxide) (1) to yield the ZrO₂layer when deposition was followed by mild thermolysis; the SAMP wasthen formed through immersion in an ethanol solution of1,4-diphosphonobutane (this step also removed remaining photoresistexposing the underlying substrate). The synthesis of SAMP/ZrO₂/substrate(4) is outlined in FIG. 1B. Eleven striped patterns defined as the widthof the SAMP/ZrO₂ stripe (in μm) X spacing between stripes (in μm), e.g.,20×30 were used.

Spectroscopic analysis was used to determine elemental composition anddistribution of photolithographic patterns of SAMP/ZrO₂ onoxide-terminated Si (SiO₂/Si), PEEK, PET, and nylon 6,6. XPS analysis ofa 30×30 striped pattern of the SAMP/ZrO₂/SiO₂/Si (4) showed peaks withbinding energy (BE) Zr(3d_(5/2))=183.4 eV and BE P(2p)=134.3 eV, withrelative integrated areas Zr:P≈1:1.5. Zirconium, P, C, and Si weremapped using SEM with EDS for a nominal 20×30 pattern on silicon, whichshowed conformity of Zr, P, and C with the pattern and withconcomitantly reduced Si signal intensity (FIG. 2); the P(K_(α))emission peak (2.013 keV) somewhat overlaps with the stronger Zr(L_(α))peak (2.042 keV). Polymer surface characterization was performed on 60×SAMP-patterned surfaces using the same techniques.

AFM analysis of striped patterns of SAMP/ZrO₂/SiO₂/Si showed heights tobe 10-70 nm; these nanoscale thick patterns should not affect physicalproperties of the substrate. AFM images of the SAMP/ZrO₂ pattern on PETshowed an average height of 70 nm. Variations in height measured on anysubstrate surface are likely due to small changes in vapor-phasedeposition conditions for 1.

Example 3 Stability of Patterns on Substrates

All surfaces maintained the ZrO₂ stripes intact for the duration of an18 day study in which they were immersed in standard cell culture medium(10% calf serum in DMEM) at 37° C. Optical microscopy showed no evidenceof stripe peeling or delamination. XPS analysis of coupons showed thepersistence, but with signal attenuation, of the Zr 3d peak and with theappearance of an N 1s peak and higher binding energy shoulders on the C1s peak These spectroscopic changes are attributed to serum proteinadsorption onto the ZrO₂. Attenuation of the Zr 3d peak was somewhatmore pronounced on the glass substrate, and it could not be detectedafter day 9. AFM analysis showed that the height of the patterned ZrO₂stripe on Si (relative to that of underivatized regions) remained nearlyconstant following an initial increase in height within 9 days ofimmersion. For example, the initial pattern height of ZrO₂/SiO₂/Si was12 nm; after immersion in culture medium this height increased to 20-25nm and remained at this level for the duration of the study from day 3to day 18. The ZrO₂/glass pattern height (before adding protein) wasalso 12 nm; after immersion it increased from 26 nm (day 3) to 50 nm(day 18). EDS analysis of the ZrO₂/SiO₂/Si pattern at day 3 showed, inaddition to the striped pattern for Zr (as in FIG. 2 above), thatnitrogen-containing material covered the entire surface.

Example 4 Cell Attachment, Spreading, and Orientation on PatternedSubstrates

NIH3T3 fibroblasts were plated on SAMP/ZrO₂-patterned or unpatternedsilicon surfaces and allowed to attach and spread for 3 hrs. Medium wasreplaced with DMEM/10% serum and, at 24 hr, cells were fixed and stainedto visualize cell shapes and alignment (FIGS. 3A-3D). The cells attachedpreferentially to the SAMP/ZrO₂ patterns and nuclei were located almostentirely on the SAMP/ZrO₂ stripes. On closely spaced patterns (separatedby 10 μm) cells were able to cross unpatterned areas and spread alongadjacent SAMP/ZrO₂ stripes.

Fluorescent images of fixed and stained cells recorded 24 hr afterseeding on SAMP/ZrO₂/SiO₂/Si patterned surfaces were used to determinethe extent to which the cells conformed to the underlying pattern.Results of fast Fourier transformation (FFT) (X. Fu et al., Tissue Eng.Pt. A 2012, 18, 631) analysis of cell images (shown in FIG. 3A-3D) areplotted in FIG. 4. Plots show the intensity of fluorescence signal perpixel (but not cell alignment) vs. angle of orientation with regard to adefined axis. The full-width at half-maximum values (FWHM) of thesedistributions in degrees of rotation with regard to this axis provide aqualitative measure of cell conformity to the SAMP/ZrO₂ pattern: LowerFWHM values indicate better conformity to the pattern. Averages of threeimages gave FWHM values ranging from 27° for the 20×30 pattern to 49°for the 100×40 pattern.

A complementary analysis was done of cell aspect ratio (the ratio ofcell width to length; an aspect ratio of 1.0 means the cell is perfectlyround) and orientation of the cell long axis with regard to the pattern(FIG. 2A-2C). The “box-plots” represent the distribution of cell aspectratio measurements (FIG. 2B). Analysis of variance for cell aspect ratiofor all groups (patterns and control) found statistical difference(p<0.001) of at least one group to the others, and pair-wise analysisfound all groups to be different from the control (p<0.0001), except the100×40 pattern (p=0.0184). As shown in FIG. 2B, cells are more elongatedon the narrower 20×10 and 30×30 patterns compared to unpatterned or100×40 patterns. The distributions calculated for the 100×40 pattern(with the largest SAMP/ZrO₂ width studied) and the control substantiallyoverlap. These results indicate that all of the SAMP/ZrO₂ patterndimensions, with the exception of the 100×40 pattern, can influence cellshape to be statistically more elongated compared to an unpatternedcontrol surface.

Cell alignment with the pattern direction was determined by measuringthe angles of cellular long axes relative to the patterns. Analysis ofvariance found a statistical difference among the individual groups(p<0.001, α<0.05), and pair-wise comparison tests found statisticaldifferences between each of the patterned surfaces compared to thecontrol (p<0.0001, α<0.0045) (FIG. 2C). In other words, all of theseSAMP/ZrO₂ stripe dimensions can influence the direction of the cellularlong axis in such a way that attached cells are oriented in the patterndirection compared to cells on an unpatterned surface. The 100×40pattern can cause alignment of cells in the direction of the pattern butdoes not cause the cells to become more elongated than does anunpatterned surface. The 20×20 and 30×30 dimensions had the narrowestdistributions for cell long axis orientation and pattern correspondence.

Example 5 Cell Proliferation on Patterned Substrates

After one day in culture SAMP/ZrO₂-treated substrates had 34±6 cells.Cell numbers increased on these surfaces over the next 3 days, to 134±32cells; this corresponds to doubling times of 1.0 day. This is typical ofcell growth on standard culture materials. Cell-spreading areas followedthe same trend: after 1 day (3017±1830 μm²), and at the end of the 3-daystudy (2912±1643 μm²) for SAMP/ZrO₂-treated substrates. These data showthat SAMP/ZrO₂-modified surfaces are not cytotoxic.

Example 6 Alignment of Cells on Patterned Substrates

Alignment of 3T3 Fibroblasts

The alignment of 3T3 fibroblasts on the foregoing patterns wasmaintained over 8 days; the cells grew to confluence and covered theentire 1 cm×1 cm silicon coupon. In this study 3T3 cells were plated on10×10 and 30×30 stripes and on unpatterned control surfaces; time pointswere taken after 24 hr, 3 days, and 8 days with immunostaining of actin(FIG. 5A-5I). At 24 hr, 3T3 cells were aligned on the 30×30 stripes, andwere oriented in the direction of the 10×10 pattern in which a singlestripe is too narrow to entirely contain a cell (FIG. 5A, D). After 3days, the cells on 30×30 and 10×10 surfaces had proliferated to covermost of the SAMP/ZrO₂ stripes and were oriented in the pattern direction(FIG. 5B, E). Cells on the control surface had grown to confluence andwere randomly oriented (FIG. 5G, H). By day 8, cells had grown toconfluence on both 10×10 and 30×30 SAMP/ZrO₂-patterned surfaces andremained aligned in the direction of the underlying chemical pattern(FIG. 5C, F). Since no physical channeling or barriers were used and theSAMP/ZrO₂ pattern is very thin (no more than 70 nm high), cells are freeto spread across the less adhesive stripes and can form a confluentlayer.

Alignment of MSCs

Similar experiments were performed with human bone marrow-derived MSCson a representative 30×30 pattern or on an unpatterned surface as acontrol on SiO₂/Si. Alignment of MSCs was examined by immunofluorescencestaining of the actin cytoskeleton after 1, 3, and 8 days (FIG. 6A-6F).After 24 hr the MSCs were aligned with and spread on the pattern surfacewith some cells spread across two stripes. Stem cells on the unpatternedcontrol were well spread but randomly oriented (compare FIGS. 6A and6D). As cells grew to confluence, they remained aligned with the pattern(FIG. 6B, C) in contrast to cells on the control surface which wererandomly spread (FIG. 6E, F). Therefore, MSCs and fibroblasts displayeda similar response to a 30×30 patterned substrate.

Example 7 Patterning on Polyester, Polyamide, and PEEK

Photolithographic patterning was extended to polymers that arerepresentative of three classes of biomaterials, polyesters (PET),polyamides (nylon), and polyetheretherketone (PEEK), which may be usedfor biomedical purposes as implantable devices or tissue scaffolds. Thesame vapor phase synthetic procedure used for surface patterning Sidescribed hereinabove was applicable to these polymer materials (FIG.7A, D, G). NIH 3T3 fibroblasts plated on 30×30 patterned SAMP/ZrO₂polymer surfaces adhered to and aligned with the pattern with conformitycomparable to that which was found for these dimensions on SiO₂/Si(FIGS. 7A-7I).

Example 8 Production of Aligned ECM on Polymer Substrate

The polyethylene terephthalate (PET) sheet with thickness of 0.05 mm wasused as received (Goodfellow Corp., Oakdale, Pa.). The sheet was cutinto ˜0.5 cm×0.5 cm square pieces with a notch in the upper right cornerto identify the top side of the PET film. The cut sheets were cleaned bysonication in isopropanol for 15 minutes, dried in a stream of nitrogengas and warmed for 10 minutes before spin-coating at 3000 rpm withdiazonaphthoquinone sulfonic ester positive photoresist (AZ5214-E). Theresist was cured for 45 seconds and exposed to UV light through aphotolithographic mask. Finally, the film was developed intetramethyl-ammonium hydroxide solution (AZ312 MIF) to dissolve away thephotoresist from the UV-exposed regions to provide 10×10, 30×30 and60×30 patterns

PET films micro-patterned with photoresist were placed in a glasschamber with inlets for vacuum and vapors of zirconiumtetra(tert-butoxide), Zr(OBu^(t))₄ (Sigma-Aldrich). The vacuum inlet wasopened to evacuate the chamber to 10⁻³ torr. Both the inlets were openedfor 5 minutes for vapor deposition under vacuum flow followed by closingthe vacuum inlet for 5 minutes to allow deposition without externalevacuation. The samples were heated to 47 deg C. using a heating tape toallow formation of surface-bound metal oxide ZrO₂ layer. The heatingtape was removed and the chamber was cooled to room temperature. The PETfilms were then immersed promptly in 1 mM solution of 1,4-butanediphosphonic acid (Acros Organic) solution in ethanol for 17 hours. Thepatterned films were sonicated for 3 minutes, rinsed with isopropanoland dried in a stream of nitrogen gas.

NIH3T3 (ATCC) cells were cultured in DMEM supplemented with 10% bovinecalf serum. The cells were maintained at 37° C. in a humidifiedincubator containing 5% carbon dioxide. NIH3T3s were trypsinized every3-4 days and were re-plated for the next passage.

Before seeding, the PET sheet was disinfected using 70% ethanol for 20minutes, washed three times with sterile phosphate buffered saline (PBS)and immersed in 1 ml DMEM in a 24-well plate. NIH3T3s were lifted fromthe culture plate using trypsin and suspended in media supplemented with10% serum to inactivate the trypsin. The cell suspension was centrifugedand the cell pellet was re-suspended in media without added serum. Thecells were counted using a hemocytometer and the PET sheet was seededwith 50,000 NIH3T3s. The cells were allowed to adhere to PET substratein absence of serum proteins for 4 hours at 37° C. After adhesion,NIH3T3s were cultured in media with 10% serum and the media wasrefreshed on day 3, 6 and 8. The media was supplemented with 50 μg/mlascorbic acid on day 6 and 8 to augment collagen type I synthesis. Thephase contrast images of NIH3T3 cells growing on PET were taken using aNikon Eclipse TS100 microscope and a Cooke SensiCam QE High Performancecamera. Cell alignment on the patterned PET is depicted in FIG. 8.Fibronectin immunostaining and fast Fourier transformation analysisconfirmed that cells and fibronectin aligned with the pattern.Fibronectin alignment and quantification of alignment of cells are shownin FIG. 9. As shown in FIG. 10, fibronectin alignment correlated withthe pattern dimension, with decreasing conformity as the patternedstripes become wider. As shown in FIG. 11, collagen type Iimmunostaining and fast Fourier transformation analysis demonstratedthat collagen also aligned with the patterns, demonstrating thatcollagen alignment can be controlled by a chemical pattern on thesubstrate.

The PET substrate with adhered cells was decellularized by treatment toremove calcium by chelation to loosen cell attachments followed byincubation with non-ionic detergent in a hypotonic buffer at alkaline pHto lyse cell membranes and solubilize intracellular components (Mao andSchwarzbauer (2005) Matrix Bio. 6:389-99). The treatment leavesextracellular matrix aligned with the pattern. FIG. 12 showsextracellular matrix on patterned PET surfaces before and afterdecellularization, fibronectin (FN) alignment and collagen type I(Col-I) alignment after decellularization, and a scanning electronmicrograph (SEM) of the entire matrix. As shown in FIG. 12, thealignment of the matrix fibrils is maintained after decellularization.The histogram and graph show that angular alignment of collagen on thepattern is good (a relatively narrow angular distribution), but thatthere is essentially no directionality of collagen on the unpatternedsurface (the very broad “peak” on the lower portion of the graph).

Primed PC12 cells (surrogates for neurons) were seeded on thedecellularized matrix and cultured in differentiating media for 72hours. As shown in FIG. 13, neurites from plated PC12 cells were alignedwith the pattern, while there was no alignment on the unpatternedcontrol.

Example 9 Patterning of ZrO₂ and ZrO₂/SAMP

Striped patterns of 30 μm in width spaced by 30 μm (30×30) were preparedon nylon, PET and PEEK treated with ZrO₂ with or without furthertreatment with bisphosphonate SAMP as described in the previousexamples. For substrates treated with ZrO₂ alone, the surface wasexposed to vapor of the zirconium alkoxide precursor of ZrO₂ for 5minutes (ZrO₂ 5 min) or 10 minutes (ZrO₂ 10 min) before heating to makethe oxide. The longer the time of initial exposure, the thicker thelayer of oxide that formed. The ZrO₂ 5 min patterned substrate was usedfor further treatment with bisphosphonate SAMP. NIH3T3 fibroblasts(30,000 cells/well) were plated on the treated substrates and imagedafter one day (FIG. 14) and 3 days (FIG. 15). As shown in FIGS. 14 and15, there was significant cell alignment using ZrO₂ alone and very goodcell alignment on SAMP-treated substrates.

Example 10 Formation of Decellularized, Cell-Assembled, Aligned ECMPatterned Polymer Scaffolding

Volatile zirconium tetra(tert-butoxide) was condensed from the vaporphase onto a pat-terned substrate; it was then heated to generatecross-linked ZrO₂ that was bound to the polymer surface throughcoordination of its ester oxygen groups to the metal centers, which wassubstan-tiated by angle-dependent XPS analysis of the interface. Then,the substrate was dipped into a solution of the phosphonic acid to formthe polymer-attached ZrO₂/SAMP, which enjoys a long shelf life atambient temperature (see FIG. 16). In this way patterns of 10 μm widestripes of ZrO₂/SAMP and 10 μm wide gaps (10 μm×10 μm pattern) wereprepared on the poly(ethylene terephthalate) (PET) of Example 8, apolymer that is used in vascular graft biomaterials and is replete withester carbonyl groups. The ZrO₂/SAMP-terminated substrate supported celladhe-sion and spreading (FIG. 17A). NIH3T3 fibroblasts on the 10×10 μmstriped pattern spread in alignment with the pattern (FIG. 17B). Becausecells were larger than 10 μm wide, they tended to spread over two ormore stripes, but in many cases one edge of a cell was aligned with theedge of the pattern. The cells remained aligned with the pattern as theygrew to confluence (FIG. 17C).

Fibroblasts plated on patterned substrates assemble a natural fibrillarmatrix containing fibronectin. During growth of NIH 3T3 fibroblasts for10 days on 10 μm×10 μm stripe-patterned PET, a robust ECM was assembled.Immunofluorescence detection with anti-fibronectin antibodies showedmatrix fibrils that were oriented with the pattern (FIG. 18B) incontrast to the matrix on the unpatterned surface (FIG. 18A). The degreeof matrix alignment was quantified by performing two-dimensionalfast-Fourier transform analysis (FFT) of the images. The radialsummation of pixel intensity in the FFT output for each angle wasplotted between 0 to 180°. A peak centered at 90° for the patternedsubstrates indicated alignment of matrix fibrils parallel to underlyingpatterned stripes (FIG. 18C). No peak was obvious for matrix on anunpat-terned substrate. The variation in height and shape of the majorpeak is represented quantitatively by full width-half maximum (FWHM)values. A significantly lower FWHM for the patterned sample indicateshigher alignment of fibrils compared to the random fibrils on theunpatterned substrates (FIG. 18D). In addition to fibronectin, thematrix also contained type I collagen fibrils (FIG. 18E) that wereco-aligned with the fibronectin fibrils and the pattern (FIG. 18F).

A mild cell-lysis protocol was used to decellularize the matrices thatcell-assembled on PET. This procedure maintains the organization anddimensionality of the matrix. Compared to cells on a two-dimensionalfibronectin-coated surface, cell activities such as adhesion, migration,and matrix assembly are enhanced when cells are grown on adecellularized three-dimensional ECM as used here. Immunofluorescenceand FFT analyses of fibronectin and type I collagen matrix fibrils afterdecellularization confirmed the matrix remained attached to the PETsubstrate and fibril alignment was maintained with the pattern (FIGS.19A-19C). Quantification showed significant alignment of type I collagenfibrils in decellularized matrix on patterned PET compared tounpatterned surfaces (FIG. 19D, E). Analysis of decellularized matrix byscanning electron microscopy (SEM) highlights the density of fibrils andthree-dimensionality of the matrix (FIG. 19F).

Example 11 PC12 Culture on Patterned Polymers with Cell-Assembled,Aligned ECM

PC12 cells, as surrogates for neurons, were plated on this alignedECM/PET material of Example 10 and were stimulated for neurite outgrowththat was quantitatively evaluated through directional analysis (FIG.20). The cells cultured on patterned or unpatterned, decellularizedmatrix extended neurites, which were visualized by staining the actincytoskeleton (FIG. 20A, B). Co-staining of matrix fibrils indicatedcorrespondence between the direction of neurite outgrowth and theorientation of fibrils (FIG. 20C, D). Measurements of neurite projectionangles relative to the patterned matrix showed the majority of neuritesin line with the 10 μm×10 μm pattern (90° peak, FIG. 20F) compared tothe lack of a single peak in neurite angles on the unpatterned matrix(FIG. 20E). The aligned orientation of neurites is also reflected in thethree-fold decrease in FWHM value compared to the unpatterned substrate(FIG. 20E, F). In this way, the cell-assembled aligned ECM supportsneurite outgrowth that is oriented in register with the underlyingZrO₂/SAMP-patterned surface. This system therefore provides a platformfor studying the effects of alignment in devices used for nervereconstruction.

Example 12 Radial Glial Cells on Decellularized, Cell-Assembled, AlignedECM Patterned Polymer Scaffolding

Radial glial cells (rat radial glial cell line C6 modified to expressGFP obtained from Dr. Martin Grumet at Rutgers University) were platedon the patterned PET material of Example 10, were attached to and werealigned to the patterns and proliferated in alignment with the patternsuntil reaching confluency on polymers.

The same radial glial cells plated on decellularized ECM adhered inalignment with the matrix fibrils and continue to proliferate for 7-10days in alignment with ECM until reaching confluency on decellularizedpatterns. Alignment of cells was quantified by direct measurements ofcell angles relative to the patterns (FIG. 21).

Radial glial cells were also plated onto decellularized matrix andcultured for variable periods of time (1-7 days) while maintainingalignment with matrix fibrils. PC12 cells were then plated onto radialglial cell/matrix substrate and co-cultured. Both cell types wereobserved to attach and grow in register with the aligned matrix on thepatterned polymers. Analysis through FFT was done showing that allneurites and glial cells were highly aligned with pattern and ECMdirection (FIG. 22). In co-culture with glial cells, neuron cells extenda dense network of aligned neurites spanning the decellularizedmatrix/polymer substrate.

Example 13 Schwann Cells on Decellularized, Cell-Assembled, Aligned ECMPatterned Polymer Scaffolding

Schwann cells (rat neonatal Schwann cells) were plated on the polymerpatterns of Example 10 and attach and grow in alignment with patternedstripes. Cells were able to grow over a 10-day period on patternedpolymers and retain alignment. Schwann cells plated on cell-assembled,aligned decellularized matrix were attached in alignment with matrixfibrils and proliferate to confluence.

Schwann cells were able to proliferate extensively on decellularizedmatrix while retaining alignment with matrix fibrils. Schwann cells inco-culture with fibroblasts on the patterned PET of Example 10 growaligned with patterns and assemble a dense ECM network of fibronectin,collagen, and other ECM proteins produced by fibroblasts and Schwanncells.

Example 14 Neurite Outgrowth and Directionality

Sympathetic cervical ganglia were plated onto substrates to assessneurite outgrowth and directionality. Substrates included a glasscoverslip coated with a 10 μg/ml solution of laminin (Laminin),decellularized fibroblast ECM on an unpatterned glass coverslip(Unpatterned ECM), and decellularized aligned ECM on a 20 μm×20patterned PET surface prepared according to Example 10 (Patterned ECM).See FIGS. 23A and 23B. A single ganglion with extended neurites is shownin each image. The substrate is indicated above the image. In FIG. 23Bneurite outgrowth was fitted with an ellipse (lines on images) todetermine if neurite extension deviated from circular, i.e., showeddirectionality. Samples on unpatterned ECM, patterned ECM, and lamininwere measured. The aspect ratios (long axis divided by the short axis)displayed in the graph were calculated and averaged for a minimum of 3samples per condition, and show that the neurites grown on patterned ECMsubstrates showed significant directionality, i.e., the aspect ratio issignificantly different from 1.0 (which would be circular or round).

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments or examplesdisclosed, but it is intended to cover modifications that are within thespirit and scope of the present invention.

What is claimed is:
 1. A neural tissue scaffold comprising a base layerand a metal oxide layer, wherein the base layer and the oxide layercomprise a pattern of oxide layer stripes on said base layer, andfurther comprise a plurality of neural cells on the stripes; whereinsaid oxide layer stripes are deposited across a tissue growth surface ofsaid base layer, with the continuous length of each stripe layerrelative to the width being sufficient to direct the outgrowth of neuralcells aligned parallel to the lengthwise direction of said stripe, andwith the underlying base layer surface exposed therebetween incontinuous parallel stripes to provide an alternating pattern of metaloxide and base layer stripes; wherein said scaffold further comprises anextracellular matrix (ECM) layer comprising fibronectin assembled by aconfluent fibroblast cell layer, where the ECM is oriented across thepatterned surface so that ECM fibrils are aligned parallel to thelengthwise-direction of the underlying metal oxide stripes and exposedbase layer stripes therebetween; wherein the ECM is decellularized;wherein said neural cells are attached to the decellularized ECM layer;wherein said neural cells are selected from the group consisting of stemcells, oligopotent cells, differentiated neurons, differentiated glialcells and neural crest cells; and wherein said neural cells form aconfluent monolayer across the ECM layer while maintaining alignmentparallel to said alternating pattern of oxide and base layers stripesand ECM.
 2. The tissue scaffold of claim 1 further comprising anon-biologic cell adhesive layer disposed on the oxide layer.
 3. Thetissue scaffold of claim 2 wherein the cell adhesive layer comprises aphosphonate.
 4. The tissue scaffold of claim 1 wherein the base layer isa soft polymeric surface where the polymer has a Young's modulus rangingfrom about 0.2 to about 100 kPa.
 5. The tissue scaffold of claim 4wherein the polymer has a Young's modulus ranging from about 1 to about30 kPa.
 6. The tissue scaffold of claim 1 wherein the base layer isselected from the group consisting of polyesters, polyamides,polylactones, polyacrylamides and polysiloxanes.
 7. The tissue scaffoldof claim 1 wherein the base layer is selected from the group consistingof silk and collagen.
 8. The tissue scaffold of claim 1 wherein the baselayer is selected from the group consisting of nylon, polyethyleneterephthalate, polycaprolactone fumarate, polyethylene glycol fumarate,and methylene bis acrylamide.
 9. The tissue scaffold of claim 1 whereinthe stripes are about 5 μm to 100 μm wide and spaced about 5 μm to 100μm apart.
 10. The tissue scaffold of claim 9 wherein the stripes areabout 5 μm wide and spaced about 5 μm apart, or about 10 μm wide andspaced about 10 μm apart, or about 20 μm wide and spaced about 20 μmapart, or about 30 μm wide and spaced about 30 μm apart, or about 20 μmwide and spaced about 10 μm apart.
 11. The tissue scaffold of claim 1wherein the metal oxide is zirconium oxide.
 12. The tissue scaffold ofclaim 1 further comprising glial support cells.
 13. The tissue scaffoldof claim 12 wherein said glial support cells are selected from the groupconsisting of CNS macroglial cells and PNS macroglial cells.
 14. Thetissue scaffold of claim 13 wherein said CNS macroglial cells areselected from the group consisting of astrocytes, astrocyte precursorcells, oligodendrocytes and oligodendrocyte precursor cells.
 15. Thetissue scaffold of claim 13 wherein said PNS macroglial cells areselected from the group consisting of Schwann cells and Schwannprecursor cells.